The Microbial Quality Aspects and Decontamination Approaches for the Herbal Medicinal Plants ...

24 Pages·2016·1.89 MB·English

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Current Pharmaceutical Design, 2016, 22, 4264-4287
REVIEW ARTICLE

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The Microbial Quality Aspects and Decontamination Approaches for
the Herbal Medicinal Plants and Products: An in-Depth Review (cid:8)(cid:2)(cid:3)(cid:5)(cid:12)(cid:6)(cid:4)(cid:13)(cid:14)(cid:7)(cid:5)(cid:9)(cid:15)(cid:6)(cid:10)(cid:7)(cid:11)
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Daniela Dal Molim Ghislenia, Marina de Souza Bragaa, Irene Satiko Kikuchia, Mirela Braşoveanub, Monica
R. Nemţanub, Kamal Duac and Terezinha de Jesus Andreoli Pintoa,*
aDepartment of Pharmacy, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, Rua Professor Lineu Prestes
05508-000, Cidade Universitária, Brazil; bNational Institute for Lasers, Plasma and Radiation Physics, Electron Accelerator Labora-
tory, 409 Atomistilor St., P.O. Box MG-36, RO-077125Bucharest-Măgurele, Romania; cSchool of Pharmacy and Biomedical Sci-
ences, The University of Newcastle, Newcastle, Callaghan NSW 2308, Australia
Abstract: Background: The present review article provides an overview of the published
literature concerning microbial quality of medicinal plants and products and their decon-
tamination methods. It is important to analyze different aspects regarding the cultivation,
growing, harvesting, storage, manufacturing, and decontamination of medicinal plant prod-
ucts. Herbal medicinal plants bear a massive microbial load leading to contamination and
mycotoxin, which needs to be considered, and properly controlled using suitable sterilization
A R T I C L E H I S T O R Y
and decontamination methods. Methods: The main focus of this review is on the definition,
advantages, disadvantages and applications of decontamination methods, particularly to
Received: June 5, 2016 show that one must consider the characteristics of the initial sample to be decontaminated.
Accepted: June 21, 2016
Results: The effects of various methods (ozone, plasma, irradiation) on medicinal herbs and

products treated for microbiological decontamination are dependent on factors related to
DOI: 10.2174/1381612822666160623 microbial load (i.e., nature and amount of initial contamination), herb/product matrix (i.e.,
070829 complexity of chemical composition, physical state - solid or liquid) and treatment conditions (i.e., time, irradia-
n
tion dose, absence or presence of oxygen). In addition, it is important to accept some loss of the chemical com-
g pounds, while decreasing microbial load to acceptable limits according to official herbal pharmacopoeias and
literature, thus ensuring a final product with quality, safety and therapeutic efficacy. Conclusion: The conclusion,
i which comes from this contribution, is that herbal medicine has more contaminants than a chemically well-
s defined drug, thus, good manufacturing practices should be followed.
e Keywords: Herbal medicinal plants, herbal medicinal products, microbiological contamination, bio-burden, quality, decontamination, GMP.
D
INTRODUCTION maintain their chemical and biological properties. The purified

l According to the World Health Organization (WHO) “a me- active constituent is then mixed with suitable solvent and excipients
a dicinal plant is any plant which, in one or more of its organs, con- in order to have appropriate shelf life and effective therapeutic ac-
tains substances that can be used for therapeutic purposes, or which tivity (i.e. the ability of the medicinal compound in the prevention,
c are precursors for chemo-pharmaceutical semi synthesis” [1]. The diagnosis and treatment of various disease states). If any synthetic
i medicinal plants can either be a wild or a cultivated plant specifi- compound is being included in a finished herbal product, it will not
t cally for the medicinal purposes. fall in the category of herbal product [3, 4].
u
The usage of the medicinal plants is witnessed from a long era
As there are extensive herbal medicines available in the present
e era, the similar material can be classified in numerous forms such and is considered as one of the important components of the tradi-
tional medicine system particularly Ayurveda. The usages of herbal
c as a powered plant material can be considered as both the herbal
products are quite popular and in demand at various low and middle
material and/or herbal preparation and when it is available in the
a form of a pack material, it is considered as herbal medicinal product class countries; as the herbal constituents are considered as “natu-
ral” or “harmless”. Keeping the fact in mind of increased awareness
m [2]. In all the herbal preparations, it is easy to standardize in terms
of safety around the globe, it became necessary to have a strict con-
of the defined amount and concentration of the active ingredients, if
the therapeutically active constituents have been identified and are trol on the herbal products usage particularly for the medicinal pur-
r poses [3, 5]. Around 25% of the prescription drugs are originated
well known. The herbal medicines are a broader term which em-
a from the plants. In specific, 121 active constituents of various
braces herbs, herbal preparations and materials including the fin-
h ished herbal product [3]. The preparation of the herbal products plants are in application for the preparation of various products. On
the WHO essential medicine list, 252 drugs are listed and out of
involves series of steps and stages that includes dicing the plant
P material to have maximum surface area while going through an 11% of these originated from the plant sources [6, 7].
appropriate and suitable extraction process followed by the frac- The increasing consumption of natural drugs has made their use

tionation and purification of the active constituents so as to a public health issue, due to the possibility of accessing products
t
without quality. The efficiency and safety must be validated
n
through ethno pharmacological surveys, documentation and techni-
*Address correspondence to this author at the Department of Pharmacy,
e cal and scientific quality to be achieved by controlling the raw ma-
Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo,
r Rua Professor Lineu Prestes 05508-000, Cidade Universitária, Brazil; terials, finished products, packaging materials, pharmaceutical for-
r Tel: [phone] (Extn: 8762) Fax: [phone]; mulation and stability studies [8, 9].
E-mail: [email protected]
u
C 1873-4286/16 $58.00+.00 © 2016 Bentham Science Publishers
The Microbial Quality Aspects and Decontamination Approaches Current Pharmaceutical Design, 2016, Vol. 22, No. 27 4265
In accordance with the European Medicines Agency (EMA), by large number of pharmaceutical industries for the treatment of
between the years 2004 and 2014, there were 1319 new registra- various ailments and disorders [18].
tions of medicinal plants and 622 new authorizations to medicinal Tropical diseases (TDs) are defined as those diseases that
plants marketing. The data from National Health Surveillance
mainly occur in tropical or subtropical regions. This definition im-
Agency (ANVISA) in 2008 have shown 512 registered herbal me- plies that TDs prevail in hot, humid conditions. A special group
dicinal drugs in which 432 contains single active constituent which among TDs, designated as neglected tropical diseases (NTDs) [19].
are derived from only one species; while 80 comprises a blend of
NTDs are a diverse group of parasitic and bacterial diseases that
different herbal medicinal products [10].
cause substantial illness for more than one billion people globally.
The global herbal supplements and remedies market is fore-
The diseases that prevail in tropical and subtropical conditions in
casted to reach US$107 billion by the year 2017 according to the 149 countries and intervene costing developing economies billions
analysis of Global Industry Analysts consulting. In 2011, revenues
of dollars every year. The persistence of these diseases, mainly
reached US$5.3 billion in the US alone, and by thinking long term
found in the poorest regions of the world, is a consequence of the
according to the WHO estimates, the present demand for medicinal precarious life conditions of the population, the social disorganiza-
plants is about US$14 billion a year and by the year 2050 it would
tion and the lack of an effective political action and educational
be about US$5 trillion. Unfortunately, the number of reports of
programs, without adequate sanitation and in close contact with
people experiencing negative effects, caused by the use of herbal infectious vectors and domestic animals and livestock [20-23].
drugs, has also been increasing [11-13].
It was decided to target various tropical diseases with various
In the 3rd Iberoamerican Congress of Phytotherapy (CIAF) held ambitions like elimination programs, patient access to the medi-
in 2012 in Brazil, experts have estimated that the herbal market in
cines in a patient compliant and cost effective manner at Global
2015 should reach US$93 billion. The researchers believe that the Partner’s Meeting in 2007 [24]. This decision clearly shows the
Brazilian extracts industry is well advanced and has reached an need of the development of various affordable and easily accessible
international level of excellence, but does not benefit local biodi-
diagnostic tests and medicines to the poor population to manage
versity for the production of functional foods and medicines to the and cure veracious diseases [19].
extent that is possible [14].
Herbal medicines are well known and recognized for the treat-
This growth of the industry reflects not only increased interest ment of neglected diseases such as malaria therapy which is evident
among consumers in these products, but also implicates new regula-
from a 2015 Noble prize winner in Physiology and Medicine; TuY-
tory challenges to appropriately monitor the quality, safety and
ouyou, a pharmaceutical chemist whose research was focused on
efficacy of the herbal products, as required for the pharmaceutical various plant constituents used for the treatment of malaria. Nowa-
drugs, as well to monitor the marketplace [3, 13].
days, the multitherapy artemisinin based therapy has reduced the
The present review is focused on the recent trends with the incidence and mortality of malaria, saving millions of lives in the
herbal medicinal products in terms of their quality specifically high- world [25].
lighting the importance of factors influencing the role of good Several researchers around the world are working to discovery
manufacturing practices, quality control and assurance methods. new lead molecules to treat NDs using natural sources, such as
Finally, reduce this burden to ensure safety and efficacy of herbal
plants [26]. Plant’s secondary metabolism yields an immeasurable
products. wealth of chemical structures which has been and will continue to
be a source of new drugs, directly in their native form and after
BIODIVERSITY
optimization by synthetic medicinal chemistry [27]. Due to this,
Biodiversity plays a significant role in the wellbeing of the herbal products have become an increasingly important component
world’s population. The world health organization (WHO) reports of the health system in many countries. Medicinal plants and natu-
have shown the importance of the medicinal plants which is evident ral products have already been included in the Brazilian Public
with the figure that 80% of the world’s population segment relies Health System (SUS), demonstrating the importance of the tradi-
on the herbal medicinal product [15]. tional medicine to the country. Also, the government implemented a
The discovery of the medicinal compounds from the plant national program to guarantee safe access to medicinal plants, the
products is quite popular and has been focused on various pharma- National Program on Medicinal Plants and Phytotherapy [28, 29].
ceutical companies, keeping in consideration that the development In various developed countries, the use of herbal products is
of a synthetic compound or derivative is a tedious process which considered as an alternative to the conventional medicines [30]. The
has lesser success rate and involves a huge financial burden [16]. popularity and demand of herbal products has expanded the global
According to Newman and Cragg (2010) the utility of natural market particularly, pharmaceutical to a greater extent. Due to this
products as sources of novel structures is still up to 50% of the ap- the quality, safety and efficacy of the herbal products is always a
proved drugs during the last 30 years are either directly or indirectly matter of concern and debate.
derived from the natural products. In the cancer area, for example,
over the time frame from around the 1940s to date, out of the 175 CHALLENGES IN DRUG DEVELOPMENT AND MANU-
small molecules, 131 or 74.8% are other than synthetic, with 85 or FACTURING
48.6% actually being either natural products or directly derived Plants contain various phytochemicals that have various physio-
from them [17]. logical functions and roles. The primary metabolites are important
for the cell life while the secondary metabolites, which are pro-
Merck in 1826 launched morphine for the therapeutic purposes
as their first commercial natural product. In 1899, Bayer developed duced from primary metabolites, play a crucial role in the adapta-
tion of plants. Both primary and secondary metabolites are impor-
the first semi-synthetic drug, aspirin that was based on a plant con-
tant active pharmaceutical constituents and being employed as
stituent called salicin, which is isolated from Salix alba. After this,
a series of plant medicinal compounds were isolated including co- therapeutic agents [31-33].
caine, codeine, digitoxin, quinine and pilocarpine. Some of these The function of the phenolic compounds is involved in the syn-
compounds have been marketed and are actively in use for medici- thesis of lignin that are common in all higher plants due to odor,
nal purposes like Paclitaxel for lung, ovarian and breast cancer, pleasant flavor and color, in addition to being useful for pollination
Artemisinin for malaria and Silymarin for liver diseases. The trend and seed dispersal. Furthermore, this group of compounds is impor-
of herbal medicine research is gaining popularity every single day tant for protecting plants against ultraviolet rays, insects, fungi,
viruses and bacteria [34].
4266 Current Pharmaceutical Design, 2016, Vol. 22, No. 27 Ghisleni et al.
The alkaloids can be defined as pharmacologically active com- avoids the complexity with the synthesis of complex structures and
pounds containing nitrogen and amino acid derivatives. The role of specific stereoisomers as well as the use of toxic chemicals [39, 46].
alkaloids in the chemical defenses of plants is supported by the Also, the demand of vegetable raw material in the market, which is
wide variety of physiological effects they have on the animals and dependent on the seasonal conditions, has also recommended the
for their antimicrobial activities. Several alkaloids are toxic to in- application of cell culture techniques ensuing the quality, safety and
sects and act as a repellent to herbivores, which end up helping in efficacy of the plant material [37, 47].
the reduction of microbial contamination [34, 35].
Improvement and harmonization of regulatory affairs are re-
Considering the importance of secondary plant metabolites, quired. Also, the new trends in the use of genetically improved
alkaloids as medicinal compounds are well postulated in medicine, plants, instead of cultivated plants in the wild, will offer great ad-
cosmetics and nutraceuticals [36]. vantages as this would make possible uniformity and high quality
of the raw materials, which are key to guarantee the efficacy and
Despite major advances in synthetic chemistry, production of
safety of herbal drugs [48].
secondary metabolites from plants has been made by cultivation of
medicinal plants for long time. On the other hand, plants originating
QUALITY ASPECTS
in specific biotypes may have many difficulties to grow out of their
local ecosystems. Some common species of plants can not be grown Good Manufacturing Practice for Herbal Medicines
on a large scale due to their susceptibility to pathogens. This issue
Good manufacturing practices (GMP) are minimum technical
has led scientists and biotechnologists to consider the cell and tissue
requirements for the manufacturing of goods and are intended to
as an alternative way to produce the corresponding secondary me-
reduce risks and damage to health, to provide manufacture and
tabolites in vitro, ensuring the end product quality, safety and effi-
marketing of pharmaceuticals with quality, efficacy and safety [30].
cacy of the final product [35, 37, 38].
In general, in manufacturing of a herbal medicine, the first step
The long history of plant cell culture, starting from the begin-
ning of the 20th Century has established a tremendous baseline of is the choice of a specific medicinal plant, is made an ethno phar-
macological study. Once the plants are defined, collection site and
knowledge and technologies. In 1902, Gottlieb Haberlandt, was
postharvest processing including primary cutting are also defined.
recognized as the founder of plant cell culture science, at first he
These steps are covered by guidelines on good agriculture and col-
attempted to initiate plant cell cultures, although he was unable to
lection practices for medicinal plants or by local guidelines in each
induce cell division [38, 39]. The potential of plant cell cultures as
country [30, 49, 50].
sources of nutraceutical and medicinal compounds also has a long
history of research, dating back at least 50 years. The ability of The safety and efficacy of herbal products are determined by
single somatic cells to survive independently, divide, and ultimately their quality. For example, the quality risks such as misidentifica-
form a complete plant is a crucial feature of plant cells that has tion/mislabeling of herbal ingredients, adulterations, substitutions
enabled the development of plant biotechnology [38, 39]. and heavy metal/pesticide/microbial contaminations can be suffi-
ciently minimized with proper quality control measures of the
Modern medicine has achieved great advancement, but it is
manufacturing process [30, 49, 50].
known that the medicinal plants play an important role in global
health, as mentioned, with the wide use of derivatives of natural Every country must apply a strict quality control and assurance
product drugs [40, 41]. However, some factors may compromise measures for the plant materials used for the medicinal purposes to
the use of medicinal plants for pharmaceutical purposes, such as avoid the incidences of adulteration and poor quality herbal prod-
climate change, standardization of production, microbiological ucts and medicines. This implies to the manufacturing, imports and
contamination and even difficulty in multiplication. In this context, marketing of the herbal products. This can be done by implicating
it becomes essential to carry out further studies of pharmacological and practicing various official regulations which can be listed on
context, therapeutic and agronomic, for the large-scale cultivation the herbal pharmacopoeias or other official documents [51].
and conservation of these species [42]. The growth of herbal plants used for the medicinal purpose
Currently, the applications of biotechnology in agriculture and largely depends upon the geographical location, particularly climate
medicinal plants have been quite widespread. Kerbauy et al. de- and soil constituents which are difficult to control and in turn affect
scribes many of these applications, such as cloning, culture cells, its productivity [49].
tissues and organs, obtain haploid plants from another culture pro- Because of the inherent complexity of naturally grown medici-
duction of secondary metabolites in bioreactor, generation of soma nal plants and the often variable nature of cultivated ones, contami-
clonal variants micro grafting, technology of protoplasts and cryo- nation with toxic medicinal plants and/or plant parts and the num-
preservation [43]. ber and small quantity of defined active ingredients, the production
The culture of cells and tissues can solve or minimize points in and primary processing has a direct influence on the quality of
the systematic multiplication of elite plants by micro propagation herbal medicines. For this reason, application of GMPs in the
process. Moreover, it can be used in the production of secondary manufacturing of herbal medicines is an essential tool to assure
metabolites that are relevant from the therapeutic point of view and their quality [49].
that, by some sort of impediment, they are not synthesized [44]. The natural origin of the herbal materials implicates microbio-
Plant cell cultures can offer continuous production systems for logical contaminants. Furthermore, during the course of harvesting
high-value food and health ingredients, independent of geographi- and processing, herbal products may be especially prone to micro-
cal or environmental variations and constraints. Yet despite many biological contamination. To avoid alterations and to reduce con-
improvements in culture technologies, cell line selection there are tamination in general, a high level of sanitation and hygiene during
few commercial successes [45]. manufacture is necessary [49].
Cell culture techniques are the robust approach, which have Requirements and methods for quality control during the manu-
greater benefits as compared to the field grown medicinal plant. facturing process of herbal products are highly complicated com-
The various factors that can affect the quality of the medicinal pared with quality control of synthetic drugs. The following exam-
plants like geographical and environmental conditions use of agro- ples can contribute for this: (a) Herbal drugs are usually mixtures of
chemicals etc. can be avoided with tissue culture techniques. Such many constituents; (b) The active principle(s) is (are), in most cases
techniques require aseptic conditions minimizing the chances of unknown; (c) Selective analytical methods or reference compounds
contamination and ensuring the purity and yield of the chemical may not be available commercially; (d) Plant materials are chemi-
constituents [45]. The extraction process from the cell cultures
The Microbial Quality Aspects and Decontamination Approaches Current Pharmaceutical Design, 2016, Vol. 22, No. 27 4267
cally and naturally variable; (e) Chemo-varieties; (f) The source and tests (physico-chemical and microbiological) must be performed to
quality of the raw material are variable [52]. ensure quality, efficacy and safety of products (Fig. 1). So, whole
Moreover, herbal products are registered in various forms in chain of herbal medicines production must be planned with preci-
sion and care and GMPs must be adopted.
different countries, presenting different technical challenges for
regulatory authorities resulting in rather different regulatory re-
Microbiological Contaminants
quirements across the regions and countries [30, 53]. The sources of
raw material and the good practices of manufacturing processes are Contaminants in herbal medicines are classified into physico-
certainly the essential steps for the quality control of herbal [48]. chemical contaminants and biological contaminants. A variety of
agrochemical agents and some organic solvents may be important
The methods of harvesting, drying, storage, transportation, and
residues in herbal medicines [3].
processing (for example, method of extraction and polarity of the
extracting solvent, instability of constituents, etc.) also affect herbal Also, the microbial contamination of herbs and/or products may
quality [54]. result from improper handling during production and packaging.
The most likely sources of contamination are microbes from the
To get a quality herbal product, it is important to consider the
ground and processing facilities (contaminated air, microbes of
manufacturing process as a whole. The first step is to select the
human origin), it cannot be excluded that extraneous matter and
correct target plant, hence, trained personnel must be present in the
filth material originating from rodents, insects, inorganic source,
pre-cultivate and harvest phase. After rating, cleaning and extrac-
human excrement, animal manure, faeces used as fertilizers may be
tion methods should be applied. Identification and standardization
present in some preparations [5].
of the active ingredients is a critical step to avoid adulteration or
even the presence of toxic compounds. Drying process should con- The microbial load on the plants is the result of various factors
sider the thermostability and microbial contamination of the prod- borne from air and soil bacteria, fungi and virus (Fig. 1). These
ucts. Due to the high contamination, decontamination methods microbial loads alter the purity and quality of the medicinal com-
should be carefully thought of. As well all steps, quality control pound. The microbial load depends relatively upon temperature,
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Fig. (1). Flow chart showing the hierarchy from herbal medicines to herbal medicines products and possible pathways of contamination.
4268 Current Pharmaceutical Design, 2016, Vol. 22, No. 27 Ghisleni et al.
humidity, handling and storage practices of both the crude and count of aerobic microbes ranged between 3.8x103 CFU mL-1 (col-
processed medicinal plant materials [55, 56]. ony forming unit/mL) and 1.6x106 CFU mL-1showed a significant
The most common physicochemical contaminants from medici- increase in Acinetobacter baumannii, Escherichia coli, Enterococ-
nal plants can include radioactive contaminants; (Ce134 (cesium- cus fecali, and Pseudomonas aeruginosa in fennel and chamomile
134), Ce137 (cesium-137); toxic metals and non-metals (lead, cad- teas while the tea bags boiled at 90˚ C have shown less molds but
mium, arsenic and others); agrochemical residues, pesticides, fertil- not the spore forming bacteria [63].
izers, fumigants, residues from various solvents and undeclared Medicinal plants are frequently contaminated with toxigenic
chemical substances. These contaminants can affect the herbal ac- fungi originating in the soil. Numerous natural occurrences of my-
tive constituent’s purity and composition [5, 54, 57]. cotoxins in medicinal plants and traditional herbal medicines have
According to Kneifel et al. medicinal plants host a wide spec- been reported from various countries. In spite of the long history
and wide use of herbal medicines as foods and traditional medi-
trum of microorganisms with various individual properties and with
cines, there are few publications on their contamination with myco-
considerable differences regarding qualitative and quantitative as-
pects. The microbial load of plants is the result of a series of influ- toxins [64].
ences caused by animate and inanimate sources, and microbial con- Mycotoxins are toxic secondary metabolites produced by molds
taminants are easily transferred via air- and soil-borne vectors [56]. such as Aspergillus, Penicillium, Fusarium and Alternaria spp. [65-
The risks caused by microbiological contaminants at different 67]. Mycotoxins can be defined as ‘‘fungal metabolites that when
stages during the production of medicinal herbal drugs are shown in ingested, inhaled or absorbed through the skin cause illness or hu-
Table 1. man and animal death’’[68, 69]. Mycotoxins cause variety of toxic
results, such as carcinogenic, neurotoxic, teratogenic and immuno-
Table 1. Evaluation microbial contamination risks at different
toxic effects [70]. Mycotoxins can be formed during plant growth
stages during the production of herbal medicine [56].
(cultivation or wild growth) or during storage of herbal sub-
stance/preparation or herbal medicine products (HMPs) [57].
Production Steps RiskLevel While the presence of molds might not be correlated with the
presence of mycotoxins, there are reports of aflatoxins, Ochratoxin
Pre-cultivation No to low risk A (OTA). and fumonisins in medicinal plants, tea and other botani-
cals [71]. The most important mycotoxins are highly toxic and car-
Field cultivation High risk
cinogenic aflatoxins. There are nearly 20 different types of aflatox-
Harvest High risk ins of which the four major ones are aflatoxins B1, B2, G1 and G2.
Aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2) are produced by
Intermediate storage Low to medium risk Aspergillus flavus, while all four isoforms (B1,B2, G1, G2) are
produced by Aspergillus parasiticus [57, 64]. AFB1 is considered
Transpostation No to low risk
to be the most toxic mycotoxin.
Treatments (clean- OTA are another family of mycotoxins produced by many spe-
Low to medium risk
ing/cutting/drying/packing) cies of Aspergillus and Penicillium [72]. The major species impli-
cated in OTA production includes Aspergillus ochraceus, Aspergil-
Final product (packaged/stored) Usually no risk lus carbonarius, Aspergillus melleus, Aspergillus sclerotiorum,
Aspergillus sulphureus, Pichia verrucossum [73]. The family of
ochratoxins consists of three members, A, B, and C. OTA is the
Among the microbiological contaminants, bacterial endospores
most abundant and toxic of the three while, ochratoxin B (OTB)
and fungal spores can be regarded as the two dominating groups of
and ochratoxin C (OTC) are less important and less common [68].
contaminants associated with medicinal plants, a broad diversity of
bacterial, fungal cells and viruses can be found either in or on the Fumonisins (FB) are mycotoxins which are commonly reported
plant material, as a consequence microbial contaminations represent contaminants of corn and corn products [72, 74]. They are quite
a great problem for herbal remedies. Fungal contamination requires stable compounds and are toxic [75]. Among all the derivatives,
great attention because, among other reasons it can produce myco- fumonisin B1 (FB1) is most toxic and exists in higher concentration
toxins [56, 58]. in medicinal plants [76] and black tea [77] as compared to fu-
monisin B2 (FB2) and fumonisin B3 (FB) [78].
Spores are one of the important reasons affecting the growth of
certain gram-positive bacteria on the herbal plants. These are pri- Aflatoxins and OTA are heat stable and soluble in hydro-
marily found on the dry herbal substance and are highly resistant to alcoholic solvents. There is therefore a potential risk of carry-over
chemicals and different environmental conditions leading to patho- of aflatoxins and OTA from the herbal substance to the herbal
genicity and are the most common cause of food poisoning [55, 57, preparation or HMP which could lead to the presence of higher
59]. concentrations of aflatoxins in the herbal preparation or HMP [57].
According to the good manufacturing process (GMP), the basic The geographical origin may have a marked impact on the ex-
quality requirements to herbal preparations and chemically synthe- tent of aflatoxin formation because aflatoxin formingmolds prefer
sized products are the same, for example identity, purity and con- elevated temperatures and humid conditions. In general herbal sub-
tent determination [60]. The manufacturers should ensure the low- stances originating from plants grown in (sub)tropical climate may
est possible level of microorganisms in the raw material, finished show significantly higher levels of aflatoxins than those grown in
dosage forms and in the packaging components to maintain appro- cooler, dried climates [57].
priate quality, safety and efficacy of the natural products [61, 62]. In most of the developing countries where the good agricultural
Various research groups have investigated the types of con- practices (GAP) and good harvesting practices (GHP) are not well
taminant in certain kinds of plants. Wilson and Roberts evaluated implemented have a greater chances of toxigenic fungi contamina-
the microbiological quality of fennel, chamomile, peppermint, and tion, which originates from soil [48, 67]. Also, the transportation
fruit teas (commercially available in tea bags) [46]. The study has and storage conditions increase the risk of fungal contamination
shown the importance of temperature in regard to the existence and affecting the growth of medicinal plant products [79]. Various mi-
growth of bacteria and tea bags were boiled at the tea prepared at crobial contaminations can be categorized as primary (from micro-
67° C was not enough to kill the bacteria and the total microbial flora of the plant itself) and secondary (due to handling of the plant
material). This can be avoided by the application of proper decon-
The Microbial Quality Aspects and Decontamination Approaches Current Pharmaceutical Design, 2016, Vol. 22, No. 27 4269
tamination methods. Such contaminations affect the quality of the As per the WHO guidelines, various methods are being postu-
plant product and in turn the formulation characteristics [55, 57]. lated for the quality control of the medicinal plant products involv-
Studies have also shown that some medicinal plants exhibit ing suitable standards and controls [90]. The various physicochemi-
cal properties been emphasized include solubility, powder fineness
antagonistic properties against microorganisms. These effects are
due to the activities of certain compounds (e.g., essential oils) and and sieve size, determination of foreign matter, macroscopic and
may contribute to the improvement and to the stability of the mi- microscopic examination, thin-layer chromatography and determi-
nations of ash, extractable, matter, water and volatile matter, vola-
crobiological status. In general, the hygienic situation of medicinal
drugs is a result of an array of positive and negative effects, which tile oils, bitterness value, hemolytic activity, tannins, swelling in-
are determined by extrinsic, and/or intrinsic factors [80-82]. dex, foaming index, pesticides residues, arsenic and heavy metals.
The application of various analytical techniques is also well defin-
Microbial Quality and Quality Assessment ing in the quality control of the plant products which includes ti-
trimetric, gravimetric, chromatographic and spectroscopic tech-
Herbal substances/preparations may be contaminated with nu-
niques [90, 91].
merous species of bacteria and fungi (yeasts and molds). The con-
tent of viable bacteria, fungi and their spores, should be determined Examples of microbial tests applicable to herbal medicinal ma-
and limited in herbal substances/preparations and HMPs in accor- terials are: tests for specific microorganisms, determination of En-
dance with the provisions of the Pharmacopeias around the world terobacteriaceae and certain other Gram-negative bacteria (detec-
[57]. Recommended microbial limits to non-sterile drugs, botanical tion and quantitative evaluation - E. coli, Salmonella spp., P. aeru-
ingredients and products according to United States Pharmacopoeia ginosa, S. aureus), validation tests for specific microorganisms,
[83], British Pharmacopoeia [84], Brazilian Pharmacopoeia [85] total viable aerobic count, microbial assay for potency, tests to afla-
and WHO [3] are shown in Table 2. toxins and others. Details of all these methods can be found in in-
ternational or in locals Pharmacopoeias [90, 91].
The active constituents of the herbal plant material have higher
microbial contamination (bioburden) and it needs strict quality Specifications for the quality of some HMP ingredients are set
control and assurance to have a safe and quality product. The Euro- out in pharmacopoeias, which are recognized as official in many
pean Pharmacopoeia has stated acceptance criteria for the microbial jurisdictions. The high quality of the finished product requires not
quality, which depends upon the preparation method of the herbal only ingredients that meet pharmacopoeia standards but also de-
product. pend on such factors as manufacturing, packaging, labeling, and
importation activities that comply with current GMPs [49] pub-
To evaluate the product microbial limits and the route of ad-
lished by WHO [3, 30, 49, 90].
ministration should be considered. The nature of the test and fre-
quency vary depending on the product. Certain categories should be Govindaraghavan and Sucher national regulatory policies de-
routinely tested regarding the total microbial contamination, such as terminate the levels of allowed health claims and, in turn, the qual-
products of vegetable origin, mineral and/or animal products, as ity requirements for botanicals in different categories. Such legal
well as high water content (aqueous oral solutions, creams, etc.) diversity, especially among countries with long history of tradi-
[85]. tional use of botanicals, make the establishment of internationally
harmonized quality guidelines quite difficult [92].
The undesired introduction of impurities of a chemical or mi-
crobiological nature, or of foreign matter, into or onto a starting According to Zhang et al. standard herbal products with con-
material, intermediate product or finished herbal product may occur trollable quality can be manufactured by using modern analytical
during production, sampling, packaging or repackaging, storage or methods and pharmaceutical techniques. However, currently
transport, so the quality control in all of these steps is essential [3]. achievement in quality control of herbal medicine is regional and
partial. To achieve the overall improvement of quality, efforts
Quality Control Methods should be made to deepen methodological researches and enhance
One of the foremost factor affecting the purity and safety of regulatory in herbal medicines. Government organization should
medicinal plants include the geographical origins of the plants; assume responsibility to provide active guidance and effective regu-
which limits its variable amount of the chemical constituents and lation. Just like any other medication, those based on plants must
affecting the therapeutic activity. This leads a challenge to the qual- demonstrate quality, efficacy and safety for use [93]. Various strict
ity assurance of the herbal medicinal plants [86]. quality control and assurance protocols are implicated and followed
during the life cycle of the herbal medicinal product starting from
Currently, there is considerable variation from country to coun-
the plant cultivation, harvesting, extraction, isolation, identification
try in the quality control of herbal medicinal plants and products
of chemical constituents and design of a suitable herbal formulation
and this variation not only impacts on public health, as its contami-
till marketing [61].
nants may represent avoidable risks for patients and consumers,
also it has effects on international trade. Thus, implementation of Decontamination and Sterilization
testing for contaminants in the national and/or regional quality con-
Two linguistic terms are usually used, microbial decontamina-
trol of herbal medicines is highly recommended by WHO [3].
tion and sterilization, when referring to methods of inactivation of
The therapeutic potential of the herbal medicinal plant can be
microorganisms, especially pathogens. In this sense, the microbial
evaluated using the finger print technology, which involves both the decontamination is perceived as the elimination or reduction of
qualification and identification of the plant material. These finger microorganism level while sterilization is defined as the complete
print technology is based on the chromatographic and spectroscopic
destruction, inactivation and removal of all microorganisms [94].
principles [87]. One of the recent advance technology include high
There are many different decontamination and sterilization
performance liquid chromatography hyphenated to diode array
methods available, each one with specific characteristics and appli-
detector (HPLC-DAD) [88].
cability. In accordance with the various regulatory agencies of
All the chromatographic finger print analysis techniques have
many countries around the world, methods for decontamination of
limitations associated, which make it difficult and time consuming.
medicinal plant materials are limited. The most often used methods
To overcome the associated problems, multivariate chemometric are those based on different chemical substances that lead to dan-
methods have been introduced to have a useful data on the plant
gerous compounds and mutagenic agents; nowadays these treat-
therapeutic potential [89].
ments are considered to be less sure, prohibited in some countries
4270 Current Pharmaceutical Design, 2016, Vol. 22, No. 27 Ghisleni et al.
Table 2. Recommended microbial limits for botanical ingredients.
Recommended Microbial Limits (CFU/g)
Microorganisms Dried or powdered botanicals Powdered botanical, extracts Containing botanical ingredients
R- Aerobacteria 105 104 104
A
PHA Mold and Yeast 103 103 103
TES OEI Enterobacteria and other Gram
STACOP negative bacteria 103 Not specified Not specified
D MA
E E. coli Absence Absence Absence
T
NI
U Salmonella spp. Absence Absence Absence
Herbal medicinal products, Herbal medicinal products con- Herbal medicinal products containing
intended for the preparation of taining extracts and/or herbal extracts and/or herbal products where
Microorganism
infusions and decoctions using drugs, where pre-treatments pre-treatment would not reduce the
boiling water reduces the levels of organisms level of organism
O- Aerobacteria 107 104 105
C
A Mold and Yeast 105 102 104
M
RA
HAEI Enterobacteria and other Gram Not specified 102 104
PO negative bacteria
H P
S
TI E. Coli 103 Absence Absence
RI
B Salmonella spp. Absence Absence Absence
Herbal drugs to which hot water Herbal drugs to which cold
Microorganism Final products for oral use
extraction process extraction process
O- Aerobacteria 107 105 104
C
A
M Mold and Yeast 104 103 102
R
PHAEIA Enterobacteria and other Gram 104 103 102
N O negative bacteria
AP
ZILI E. coli 102 10 Absence
A
R Salmonella spp. Absence Absence Absence
B
Herbal materials that have been
pretreated (e.g. with boiling
Herbal medicines to which boil-
Microorganism water as used for herbal teas and Other herbal materials for internal use
ing water is added before use
infusions) or that are used as
topical dosage forms
Aerobacteria 107 107 105
Mold and Yeast 104 104 103
O Enterobacteria and other Gram
H 104 104 103
W negative bacteria
E. coli 10 102 10
Salmonella spp. Absence Absence Absence

or restricted in most advanced countries for health and environ- Complete elimination of microorganisms from a given herbal
mental safety reasons [94]. As an example, ethylene oxide (EtO) substance, preparation or HMP, by sterilization methods, is not
used as a pesticide for medicinal plant materials decontamination necessary, provided that pathogenic microorganisms are not present
has been forbidden in many countries in Europe and also in Brazil in the finished product. In addition, preventive measures are pre-
for this purpose due to the formation of toxic by-products, such as ferred rather than interventions for decreasing the contamination
ethylene chlorohydrin and ethylene glycol [57, 90, 95]. [57]. However, many researchers evaluate the application of meth-
ods in order to reduce microbial bioburden of plants and natural

products even though good manufacturing practices were adopted.
The Microbial Quality Aspects and Decontamination Approaches Current Pharmaceutical Design, 2016, Vol. 22, No. 27 4271
The application of methods to reduce the microbiological contami-
nation must be seriously studied, and the tested parameters should
be well documented and validated to ensure the quality, safety and
efficacy of the product to its final consumer [96, 97]. (cid:22)(cid:18)(cid:2)(cid:21)(cid:24)(cid:19)(cid:25)(cid:12)(cid:26)(cid:24)(cid:25)(cid:19)(cid:26)(cid:21)(cid:24)
As mentioned, sterilization is the process of destroying or re- (cid:11)(cid:12)(cid:18)(cid:19)(cid:20)(cid:21)(cid:22)(cid:23)
moving all viable life forms of microbial life, including bacterial
spores. In practice, this is achieved by either exposure to an inimi-
(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:4)(cid:7)(cid:8)(cid:3)(cid:4)(cid:9)(cid:10)(cid:11)(cid:12)(cid:6)(cid:7)(cid:13)(cid:3)(cid:14)(cid:15) (cid:24)(cid:6)(cid:27)(cid:11)(cid:12)(cid:6)(cid:7)(cid:13)(cid:3)(cid:14)(cid:15)
cal physical or chemical agent for a predetermined period or the
physical removal of organisms. The inimical agents used are ele-
vated temperature, ionizing radiation, or chemicals either as a gas,
(cid:16)(cid:29)(cid:6)(cid:15)(cid:6)(cid:29)(cid:5)(cid:9)(cid:7)(cid:8)(cid:3)(cid:4) (cid:21)(cid:28)(cid:3)(cid:4)(cid:6)
gas plasma, or a liquid [98, 99]. Physical removal involves passing
solutions or gases through a filter capable of retaining microorgan-
isms [98, 100]. Every sterilizer must meet the Sterility Assurance
Level (SAL) of 10-6 or a 12-log reduction of microbial bioburden. (cid:20)(cid:6)(cid:9)(cid:7)(cid:11)(cid:19)(cid:29)(cid:6)(cid:9)(cid:7)(cid:17)(cid:6)(cid:4)(cid:7) (cid:16)(cid:10)(cid:9)(cid:15)(cid:17)(cid:9)
With each log reduction, 90% of all microorganisms are killed; a 6-
log reduction ensures that 99.999% of microorganisms are killed.
Each spore strip used in the biologic testing of sterilizers is inocu- " (cid:17)(cid:8)#(cid:9)(cid:7)(cid:8)(cid:3)(cid:4)
lated with 1 million nonpathogenic spore-producing bacteria. Low-
temperature sterilizers use Geobacillusstearothermophilus and/or
Bacillus atrophaeus [101, 102]. $%(cid:3)&(cid:13)(cid:8)(cid:10)(cid:8)(cid:28)(cid:9)(cid:7)(cid:8)(cid:3)(cid:4)
The assurance of sterility process is provided by (a) the initial
validation of the process, (b) its periodic revalidation, and (c) the (cid:20)(cid:8)#(cid:13)(cid:11)(cid:16)(cid:29)(cid:6)(cid:15)(cid:15) (cid:29)(cid:6)
implementation of effective procedures for control and monitoring, (cid:16)(cid:29)(cid:3)(cid:31)(cid:6)(cid:15)(cid:15)(cid:8)(cid:4)#
demonstrating that the validated process has been delivered. The
validation and routine control of a sterilization process requires a (cid:26)(cid:4)(cid:15)(cid:7)(cid:9)(cid:4)(cid:7)(cid:11)(cid:2)(cid:3)(cid:4)(cid:7)(cid:29)(cid:3)(cid:10)(cid:10)(cid:6)(cid:14)
number of interrelated activities to be carried out effectively. For (cid:16)(cid:29)(cid:6)(cid:15)(cid:15) (cid:29)(cid:6)
this reason, sterilization processes are undertaken within a quality
management system as defined by the requirements for GMP or in
the international standards for quality management systems for (cid:26)(cid:29)(cid:29)(cid:9)(cid:14)(cid:8)(cid:9)(cid:7)(cid:8)(cid:3)(cid:4)
medical devices [103].
There are different methods available for sterilization and the
(cid:18)(cid:7)(cid:13)(cid:9)(cid:4)(cid:3)(cid:10)
choice of the most suitable one depends on: (a) the type of material
or substance to be sterilized, its chemical and physical properties;
(b) the purpose and the process step; as well (c) their mechanism of (cid:25)(cid:10)(cid:30)(cid:9)(cid:10)(cid:8)(cid:4)(cid:6)(cid:11)(cid:3)(cid:29)(cid:11)(cid:25)(cid:31)(cid:8)(cid:14)
action, because these sterilization methods could affect the pharma- (cid:23) !(cid:15)(cid:7)(cid:9)(cid:4)(cid:31)(cid:6)(cid:15)
ceutical formulations in different ways. Thus, the sterilization

method chosen must be compatible with the item to be sterilized to
avoid damage. Among the main sterilizing agents include: dry heat,
Fig. (2). Decontamination methods.
pressure vapor, ethylene oxide, formaldehyde, plasma, peracetic

acid, electron-beam, ozone, and gamma radiation [104].
products are listed in Table 2. A decontamination method must be
According to Chatterjee et al. herbs are susceptible to contami- chosen considering the type and composition of herbal material, its
nation with microorganisms and insect pests during processing and intended use and route of administration. Also, the storage and
storage, thus reducing the shelf life of the herbs. The microbial transportation of the herbal medicinal plant is important to taken in
contamination can also cause major illness particularly if the herbs consideration which may alter its stability and quality by various
are in contact with food poisoning organisms such as Salmonella chemical (oxidation, hydrolysis or biochemical changes due to al-
and Staphylococcus aureus. Adoption of GMP can reduce the con- terations in pH, temperature etc.) or biological phenomenas (micro-
tamination during processing and storage to a great extent. bial growth etc.) [57]. The reduction of microbial load by applying
The decontamination methods involving heat and various decontamination methods must be seriously investigated in order to
chemicals results in the reduction of the aroma of the herbal prod- guarantee a product insignificantly affected by the decontamination
ucts by attacking the heat liable components of the plant material process, the tested parameters should be well documented and the
during the process which are responsible for aroma and flavor of procedures should be validated to ensure the quality, safety and
the herbal plant but overall these techniques have effective decon- efficacy of the product to its final consumer. Fig. (2) illustrates
tamination approach [105] . some available methods of decontamination of herbal medicinal
products and traditional extraction methods herbal medicinal prod-
Decontamination process of herbal medicines may be per-
ucts.
formed by different means, as shown in Fig. (2), in spite of the
growth of industrialized forms (pills, extracts, tinctures and others). The contents and characteristics of biological active substances
Also, it should be given special attention to the changes in some such as essential oils, flavonoids, glycosides, anthocyanins, poly-
important herbal product aspects such as their microbiological, phenol acids, triterpene, saponins and oleanosidesmight be modi-
physicochemical, pharmacological and therapeutic properties is fied significantly after decontamination treatment. It is also known
generally used to reduce or eliminate microorganisms from medical that some preservation techniques appliedto herbs cause physico-
devices, surfaces and environment such that they cannot reach vul- chemical changes to these. Consequently, it is necessary to analyze
nerable sites to cause infection [100]. and certify herbal products before and/or after processing to ensure
the quality of the products for the consumers [106].
The important criteria required in the selection of the decon-
tamination methods (microbial quality and the recommended accep- Table 3 presents the various examples of decontamination
tance criteria) with regard to the non-sterile pharmaceutical methods. The main reason for employing the decontamination
methods is to enhance the shelf life of the herbal medicinal product.
4272 Current Pharmaceutical Design, 2016, Vol. 22, No. 27 Ghisleni et al.
The Committee on Herbal Medicinal Products (HMPC) has laid It is very important to have a product with good shelf life and
various decontamination methods that can be implied on herbal this can be achieved by the addition of preservatives and certain
medicinal plants. The various disinfectant methods have been dis- anti-oxidant compounds that can inhibit the fungal growth, genera-
cussed below [57]. tion of free radicals and various mycotoxin secretions. The selec-
Extraction Methods tion of preservatives and anti-oxidants would be done on the basis
of their safety and efficacy [121].
The production of a herbal medicine generally involves the
steps in which a vegetable is subjected to unfavorable conditions Essential oils are gaining attraction by various industries due to
their physicochemical characteristics like volatility and biodegrad-
for survival of a microorganisms [57].
able nature. There are almost 17.000 plant species which produces
Extraction is one of the important processes involved in the
the essential oils which have great pharmaceutical and medicinal
isolation of the active constituents from the plant product. This step
value [123].
needs special attention and it can be altered and affected by various
factors such as temperature, selection of solvent, extraction method In the past, various plant materials have found alternative ther-
apy for the infectious diseases. The main reason of having this pre-
involved, nature of drug/plant product etc. It is important to have a
proper research on the extraction method and the parameters in- servative potential in these plants has been attributed to the high
volved with it before starting the extraction process to ensure high amount of antimicrobial constituents such as flavonoids, glycosides,
alkaloids and tannins [124].
productivity and quality of the chemical constituents [107-110].
One of the most commonly employed methods of preservation
Thus, a good extraction method depends on the choice of the
includes dehydration with an aim to remove the moisture from the
solvent, process temperature and specific characteristics of active
ingredient. Adequate extraction methods help to reduce microbial plant material, which leads to the deterioration of the plant product.
Treating with the hot air is one of the way but it has its limitations
contamination also achieve microbial limits specified by pharmaco-
which brings the chemical and physical changes in the plant con-
poeias [61].
stituents affecting its quality and stability [125].
At home, dried plants can be ingested as teas (plants steeped in
hot water) or, rarely, tinctures (plants in alcoholic solutions) or Heat Treatment: Dry or Steam
inhaled via steam from boiling parts. The aqueous preparations According to Griffiths and Ribeiro, the main approach for car-
have high incidence of microbial (bacterial and fungal) contamina- rying out sterilization with the milk and dairy products include heat
tion but the preparations containing alcohol have better chances to processing where the product needs to be treated with either ultra-
remain stable for a longer duration due to the preservative proper- high temperature (UHT; 135-150˚C for 2-20 seconds) or by high
ties of alcohol. Higher ethanol concentrations (60 to 95%) have temperature short time (HSTS), which is also called as Pasteuriza-
marked bactericidal and fungicidal effects against vegetative forms, tion (-72˚C or above for 15 seconds or longer) and low-temperature
but some preservative effect is also seen at lower concentrations long-time (63˚C for 30 minutes) [126].
(above approx. 20%). Besides the ethanol concentration, the antim- Short heat treatment (ultra-high heat/ultra-heat treatment (UHT)
icrobial activity depends on the exposure time, temperature and
or pasteurization may be performed before drying herbal sub-
microbial strains present. Ethanol is ineffective against bacterial
stances/preparations/medicinal products, if necessary, in order to
spores [55, 57,111-114]. minimize microbial contamination. However, such treatments are
At present, various traditional extraction methods (maceration, not usually suitable for extracts with high contents of resinous sub-
percolation, infusion, decoction, soxhlet etc.) are still in practice stances, highly viscous extracts (dry residue more than 50%) or
due to their efficiency and reproducibility. With the advent in tech- extracts with thermolabile or volatile constituents [57].
nology, various newer extraction approaches have been in picture Heat treatments have been also developed as a non-chemical
like use of ultrasound, microwave assisted solvent extractions, method of disinfection of fresh harvest fruits and vegetables. Post-
which are robust and less time consuming than the traditional
harvest heat treatments to control decay are often applied for a rela-
methods [115]. Also, the recent advances are eco-friendly and give tively short time (seconds to minutes) because the target pathogens
better yield of the chemical constituents [116-120]. are found on the surface or in the first few layers under the skin of
One of the researches has clearly shown that the application of the fruit or vegetable. In this case, the heat treatment is being em-
various technological processes like decoction and spray drying ployed to reduce decay are fungistatic but not fungicidal [127].
reduces the microbial contamination (number of viable aerobic
Lurie, S. suggests that heat treatment substitutes a non-
microorganisms including fungi and yeast). The attempt was made damaging physical treatment for chemical prevention and there are
on the aerial parts of Phyllanthus niruri which is commercially
three methods in use to heat commodities: hot water, vapor heat and
acquired from Industry QuimerLtds, Sao Paulo [61].
hot air. Hot water was originally used to fungal control, but has
Preservation been extended to disinfection of insects. Vapor heat was developed
There are numerous attempts been made so as to preserve the specifically for insect control, and hot air has been used for both
therapeutic activity of the medicinal plant [55] and this involves the fungal and insect control and to study the response of commodities
to high temperature. Vapor heat and hot air have subdivi-
application of good agricultural collection practices (GACP) and
good manufacturing practices (GMP] with the herbal medicinal sions, sometimes air is relatively static and sometimes air flow is
plants so as to ensure their good quality and therapeutic activity [3]. quite high; additionally, hot air can have humidity control or not.
All of these permutations may affect the response of the commodity
The role of such practices will be implicated from the starting step
till the development of stable herbal product. Furthermore, the to the heat treatment and affect the length of time of exposure
shelf-life of the herbal product can be enhanced by the addition of needed to achieve a desired effect [128].
various compatible preservative [57]. According to Pushpalatha et al. pasteurization is a method of
reducing bioburden in spices and other products, which employs
Post-harvest fungal deterioration of herbal raw materials used
moist heat or super-heated steam under pressure to destroy mi-
as pharmaceuticals and nutraceuticals is a serious concern, particu-
larly in tropical and subtropical countries where the environment crobes. Moist heat kills microorganisms by denaturing cellular pro-
teins. Also, pasteurization process does not involve the use of any
hot and humid [121]. Incidences of postharvest diseases may occur
chemicals and the approach becomes more relevant in the develop-
during the different stages of the postharvest chain; during harvest-
ing, field handling, packing operations, transportation and storage ment of preservative-free formulations and can be employed with-
out any regulatory concerns for product registration [129].
[122].
The Microbial Quality Aspects and Decontamination Approaches Current Pharmaceutical Design, 2016, Vol. 22, No. 27 4273
Fumigation need to be addressed with regard to freeze-drying technique and
According to the Guide Treatment Manual from the United need detailed research [57].
States Department of Agriculture (USDA), fumigation is the tech- Freeze-dried products being hydrophilic have greater chances
nique in which the plant material is treated with a gaseous com- of having microbial growth and lesser stability when it comes in
pound which either kills the microorganisms (bacterial, fungal or contact with air but till now there is no report showing that freeze-
molds) by direct action doses [130, 131]. dried material has lesser shelf life when exposed to oxygen and
Traditionally, drying of the plant products especially the roots humidity [140]. A comparative study of three different techniques;
pulverization, extraction in liquid phase and spray drying using
and rhizomes which take longer to dry was common. Recently it
Phyllanthus niruri L. have shown that extraction in liquid process
is being replace by sulfordioxide treatment in a closed chamber
where the gas penetrates the plant material [132]. It is most com- reduces the microbial load while the other two techniques have no
effect on the microbial load [25].
monly employed for the traditional Chinese medicines (TCM)
which contains a large amount of moisture [133]. Freeze-drying is reported to decrease microbial contamination,
but there is little information on the effect of this technique, some
This practice not only reduces the drying time and prevent the
microbial growth but also imparts a pleasing white color to the herb recent results on freeze-drying for decontamination or sterilization
[132, 134]. Alternatively, the plant material can also be treated with of herbs or herbal drugs reported in literature are listed in Table 3
[57].
agents like sodium and potassium sulfite (s) to maintain its look and
color [135, 136]. High Pressure Processing
Nowadays, the sulfur fumigation is widely applied in TCM High pressure processing (HPP), also known as high hydro-
processing factories in China, however, sulfur-fumigated TCMs static pressure (HHP) processing, is a method where the material is
(SF-TCMs) have recently emerged as a controversial topic. The subjected to elevated pressures applied with a pressure transmitting
first one is consumer complaints related to the side effects of sulfur medium (water or other liquids) [57].
itself. Also, the increasing concern on the residues in SO and
2 HPP is a technology introduced in 1980s, which improves the
detrimental heavy metals in TCMs. Following, the high amounts of microbial safety and shelf life of the product [141]. It is quite com-
residues in SO2. and heavy metals and a decrease in the bioactive monly applied to the food processing industries. This is a non-
compounds revealed by a chemical analysis [136]. Despite
thermal technology that leads the destruction of undesirable micro-
prohibition of treatment with sulfur dioxide in the Pharmacopeia of organisms and inactivates enzymes without affecting the bioactive
China, farmers are still employing this approach without constituents. It also has lesser chances of losing the volatile com-
considering the associated potential toxic side effects [133].
pounds [142].
In addition, a solution of sodium or potassium metabisulfite,
It is very crucial to understand the plant structure particularly
bisulfite or sulfite can also be used as fumigation agents. Herbs can
cellular so that the modifying enzymes can be targeted which con-
also be fumigated with sulfur dioxide gas, sometimes generated by trols the various mechanisms and help in reducing the microbial
burning of bituminous coal [137].
load [143]. Therefore, it is very important to select the decontami-
Freeze-Drying nation method keeping in consideration the safety and efficacy of
the herbal products.
Drying is a process in which the moisture content of the plant is
reduced using suitable methods which helps in preventing the en- Balasubramaniam et al. have shown that HPP technique, based
zymatic and microbial activity and finally ensures a good shelf life on pressure ranging from 100-800MPa without any involvement of
of the product. It is an appropriate method for the preservation of heat can cause microbial (bacterial, yeasts, moulds and viruses)
post-harvest herbal medicinal plants. Also, it contributes to reduce decontamination. The pressure transmitting fluid is used for the
the weight and volume of the plant with positive consequences for pressure which is water for industrial scale-equipment while glycol,
transport and storage [55]. silicone oil, castor oil or sodium benzoate solution for the labora-
tory or pilot scale purposes [144].
Lyophilization or freeze-drying is commonly used in the phar-
maceutical and biotechnology industries to improve the stability of With HPP technology, the strains of microorganisms and their
formulations. The active pharmaceutical ingredient is solubilized growth phase plays vital role. Gram-positive bacteria are highly
in a solvent (usually water) and it is filled into vials, and then resistance to high pressure as compared to the gram negative. Simi-
loaded into a lyophilizer where the solution is frozen. Subsequently, larly microbial cells in the stationary growth phase are highly
it is heated at very low pressure to sublime the solvent and remove resistant to pressure as compared to the exponential phase
it from the formulation. As the moisture content is being removed [144].
from the product, it is being sealed under the vacuum and the head Georget et al. evaluated how pressure achieve inactivation of
space of the product vial is filled with an inert gas such as nitrogen
the vegetative microorganisms. They have shown that the factors
or argon due to which the formulation changes into a porous cake leading to cell death include protein unfolding or denaturation,
having extended shelf life with lesser moisture content [138]. phase transition of the cell membranes, ribosomes disintegration
Gallo et al. have demonstrated the efficiency of the spray dry- and changes in the intracellular pH leading enzymes inactivation or
ing and influence of its variables on the physical properties like damage to the membrane [145].
moisture content and flowability of herbal medicinal extract [139].
Oey et al. have highlighted the various advantages and draw-
Freeze drying approach is a dominating approach over the other backs of the high pressure technique. They have shown as com-
drying methods and most commonly employed in pharmaceutical;
pared to the thermal treatment, the high pressure treatment is al-
particularly vaccines and food industry. Recently it has shown that
ways transmitted uniformly if applied at a given position and it
freeze-drying also has some effects on the constituents of the herbal distributes in all the directions equally [146]. Although it transmits
plant material which is still under the investigations [140].
pressure uniformly; it cannot avoid the heat transfer during the
Abascal et al. concluded that freeze-drying has some unantici- building of pressure and decompression.
pated and potentially significant effects on constituent profiles and
Instant Controlled Pressure Drop
the medicinal action of plants [140]. Nevertheless, freeze-drying
In recent years a new technology, instant controlled pressure
technique is still not clear, as have certain assumptions that affect
drop (DIC for Détente InstantanéeContrôlée) has been developed as
the validity of the technique like incorporation of the cyro-
a decontamination process, particularly for heat-sensitive solids and
protectants may help in growth of microorganisms. Such issues