P. Kakkar
Scientist-F and Head, Herbal Research Section, Indian Institute of Toxicology Research, P.O. Box-80, M.G. Marg, Lucknow - 226 001, India
E-mail: pkakkar@iitr.res.in
I have been given the task of talking about the toxicological assessment of herbal materials, and also its progress in India. Now, progress in India is a very big topic, but I will try to cover whatever I can and that too in terms of Toxicological Assessment.
Let us begin with the question - Is the toxicity testing of traditionally used herbal medicines required? We must question this, because there is assured safety, as herbal medicines have been used in India for more than 4000 years as per documentation and earlier also. However, if these herbs are pharmacologically active they will have the potential to be toxic too, if used in a very high dose. Hence, toxicity evaluation is required for these materials also. It is a very systematic evaluation and we need to have our minds open when doing this toxicological evaluation.
There are two things that have happened over the past say 25 years or maybe more in India. One is, there has been a shift from individual medication by the Vaidji to larger scale production of these herbal medicines or herbal formulations. There is also an economic aspect attached to it. We would like our country to grow and reach the bar, which is 62 billion dollars, so let us not have the share of 1.5%, we want to have a better percentage in that market.
The safety evaluation of herbals would depend on two or three things; one is the purity of the preparation. If the preparation is not a standardized one, that particular individual preparation can cause toxicity. It does not mean that all such preparations are toxic. Second is, it should be free from contamination. There should not be any contaminant in the medicinal plant preparations and then a systematic toxicity evaluation is desired. The quality may be compromised due to a number of factors. One is misidentification — not many people know how to identify a medicinal plant and particularly a plant species. Then there can be adulteration, substitution because of economic reasons, because the species may be endangered, maybe cost-wise very high so substituted with another one, or not available so substituted with another one. There can be contamination, which is always inadvertent and not with any intention as such. Storage of the material and shelf life can also compromise the quality of the preparation. Quoting one study where 25 commercial Panax ginseng preparations were evaluated for seven marker compounds, they differed significantly from the ginesenosides’ content on the label, that is, 15-fold in the capsules and 36-fold in the liquid preparations, which is a very high amount of variation.
The question is, ‘do we really have standardization?’ and AYUSH took a lead in developing pharmacopeial standards, which Dr. Sharma mentioned earlier. We have been a part of this study and we have concentrated on 17 poisonous plants; altogether 24 medicinal plants were done in this and we developed the chromatographic finger print profile and also the microscopic studies to identify the plant. Seven of these monographs have already been published. We can also go in for say genomic parameters liked RAPD, RFLP, and identify of the species using that. In our laboratory, we have conducted it for Desmodium gangeticum a very important constituent of Dashamula. There can be an intrinsic toxic principle in a medicinal plant. This kind of information can come from literature and from reports, and that is why it is very important to report and document it.
One example that is given here is over 100 cases of irreversible nephropathy reported in young women attending a slimming clinic, since 1993, and some developed urothelial cancer also. There was an inadvertent use of the toxic Aristolochia fangchi root in the formulation as a substitute for Stephania tetrandra. A very recent report in 2010, mentions that consumption of aristolochic acid containing Chinese herbal products (studied in 4574 patients) was found to be associated with an increased risk of cancer of the urinary tract, in a dose-dependent manner. So, if we have this kind of information and we also have information about aristolochic acid being present in the preparation, then we can be cautious right from the beginning. There are certain warnings that keep coming from time to time like the Food and Drug Administration (FDA) issued a consumer advisory that Kava products may be associated with severe liver injury.
These types of toxic herbs may not be present in the dietary supplement or medicines, but what about topical applications. The pyrrolizidine alkaloids in Comfrey is one of the examples. Comfrey is one of the most popular herbal teas in the world, which contains pyrrolizidine alkaloids and FDA took action to remove Comfrey from all dietary supplements in 2001. But you see, there are creams and cosmetics which contain this and they are being marketed. Some of the families of medicinal plants in which pyrrolizidine alkaloids are present are Boraginaceae, Asteraceae, and Fabaceae. Pyrrolizidine alkaloid has minimum toxicity, but when it is metabolized in the liver then the pyrroles formed are toxic, so it causes liver damage. Apart from liver damage it also causes damage to other organs; it can infiltrate the lung fluids, it can cause pulmonary edema, and in high doses it can be fatal. So there are the non-target toxicities also.
That was about quality and intrinsic toxic principles that may be present in the medicinal plant and now coming to the contaminants; herbs should be free from contaminants. What are the contaminants that we should look for? Heavy metals, pesticides, microbial load, and radioactivity. Radioactive load may be related to the location, so one has to exercise one's own wisdom to see whether it is required to be tested or not. However, with regard to the other three, they do require testing. Heavy metal may be present as an ingredient, as in some of the ayurvedic preparations or as a contaminant. The commonly tested pesticides, synthetic pyrethroids, and organophosphates, have limited half-life, so they do not cause much of a problem but organochorines do cause a problem because they are residual in nature and persistent, and studies show that even 25 – 30 years after banning DDT in the US, the residues are present in the soil. So, the persistent nature of these pesticides requires that these should be tested. Now with this knowledge, in our country DDT is not allowed to be used for agricultural purposes, but it is used for vector control because we have a problem of malaria.
Hence, we took up a systematic study of heavy metals and persistent pesticides in medicinal plants. Since 2002, taking up studies in three simultaneous projects, we have analyzed more than 1800 plant samples. You can see the map and the red dots in more than 100 locations in the country from where the samples were collected, belonging to 312 plant species, and heavy metals, volatile and non-volatile; both were tested. We tested for DDT (op’ and pp’), its metabolytes, all the four isoforms of HCH and alpha endosulfan and tried to collect samples equally from each zone. If you would see, the data generated from the analysis of all the 1800 plants, for lead, cadmium, arsenic and mercury (Pb, Cd, As, and Hg), then the level of heavy metals is not very high. In our study, hardly 1.5 to 2% of the plants contained lead over the permissible level.
Also, different countries have different permissible limits. In Singapore the permissible limit for lead is 20 mg, whereas, in our country it is 10 mg. But now we have the directive that lead, cadmium, arsenic and mercury are to be tested in all herbal raw material extracts and all the finished products. Among the persistent pesticides, most of the time they were below the detection limit, but the data that you see here is just for the detected ones; although it does not mean that if it is detected, it is above the permissible limits. As we have sophisticated equipment we can detect it at a very low level.
Just a finding that some heavy metal or pesticide has been detected, does not mean that it is toxic. We have to exercise our wisdom to see what is toxic and what is not toxic. It does not have to be alarming if DDT is just detected in a number of plants. The Commerce Ministry fixed specification limits for heavy metals and pesticide residues, which came out in 2007. As we have generated a large data volume, we have developed a database for it, which has recently been copyrighted, where we have the names of the plants. It contains the plant part, it gives you information regarding the region from which it has been collected, and the date on which it has been collected, and from the data, one can generate a report according to one's own need. It will give you a picture of all the plant parts that we have analyzed.
We have also analyzed the microbial load in medicinal plants as per the WHO permissible limits, bacterial count should be equivalent or less than 105 colony forming units (CFU) and fungul count should be less than 104 CFU / g crude plant material. E. coli and Salmonella must be absent in the plant material.
Now coming to toxicity testing, it depends on the intended use. Here I am citing one example of garlic; it can be used as a food additive, conventional food, dietary supplement food, for special dietary needs, can be used as a biological drug or a medical device — some people use it as an ear plug and in some of the cosmetics. There are several toxicity testing guidelines available, for example, OECD, ICH, FDA, WHO, and so on, which outline the testing requirements. The minimum testing required is Acute toxicity: 14-day study, Sub-acute toxicity: 28-day study, and Chronic toxicity: 90-day study. The duration of the repeat dose studies depends upon the intended use of the herbal medicine (e.g., single or repeated clinical exposure for less than one week, the administration period for toxicity study is two weeks to one month; for long-term repeated administration > 6 months, the administration period for toxicity study is 9 to 12 months, and so on)
The use of animals has also been reduced. Now we have a test for acute toxicity (OECD 420) which we can do with only five animals (female rats). One animal is taken for a sighting study, with a fixed dose and observations done for 14 days for toxic symptoms and mortality. The main study is conducted with four animals. The cage side observations are done, which are routine, and the gross pathology after the sacrifice of the animals. So with just five animals you can do the fixed dose acute toxicity study for herbals, which have a history of use.
Coming to metal toxicity, it is a function of the specific metal, which metal has been used, the form of the metal that is used, whether it is a sulfide or an oxide, as also the exposure, how long has the person been exposed to it. Thus, the dosage and the duration are very important and the question to be asked is: are these heavy metals present in the herbometallic preparations? Are they bioavailable after specific processing, especially if using the herbometallic preparation, with presence of toxic heavy metals as an ingredient? Whether prepared according to the ancient text, has there been any quality control, how to assess the metal present in the desired form like oxide or sulfide or it is in some other form or is it retained by the vital body organs? As far as quality control is concerned we did a metal estimation in Tribhanga Bhasma prepared by three different Centers (this was done in a project with AYUSH). The heavy metal contents were similar in the preparations across the three Centers. So the quality is maintained if it is prepared according to the text. We were also involved in the preparation of the protocol for evaluation of herbominerals. We have done some study for coded herbometallic preparations and we found that subacute toxicity with recovery and a subchronic oral toxicity study showed no toxicity at all of these herbometallic preparations. This was an initiative to clear doubts regarding the safety of metal containing herbal preparations, because that is one line of Ayurveda which is very effective and poses a lot of questions and needs answers.
Another milestone has been the golden triangle partnership project that Dr. Vaidya has been very instrumental in bringing out. In this program, Ayurveda meets the modern medicine and the basic science. The CSIR, Department of AYUSH and ICMR together went into this project, which was started sometime in 2006, and the idea was to validate the traditional Ayurvedic drugs and develop new herbal formulations. IITR has been involved in the development of eight of the new formulations out of which dossiers have been submitted for six. For three herbomineral drugs (Ras kalpa), 90 days oral toxicity was also done and they were found to be safe. In one of these, its toxicity studies were done as well as metal content estimated in all the vital organs. When we used 10 times the therapeutic dose, there was a slight increase in the metal content, which went down during the recovery period. There was a recovery period of 30 days and after that the metal content was quite close to the level in non-treated animals. So this was an answer to whether metal was getting accumulated in the vital organs or not. It would cause toxicity if it got accumulated in the vital organs in high amounts. Some specialized tests that could be required were the immunotoxicity parameters. Here the Ayurvedic text can give you indications, because many of these side effects and other things are already mentioned there.
Specialized tests required may include immunotoxicity and allergenictiy parameters (IL1 b, IL 4, IL 6, IL 10, TH1, TH2, histamine levels, etc.), mutagenicity tests (Ames, micronucleus assays), and medium- and long-term animal tests for carniogenicity as per the international agency for research on cancer guidelines. Reproductive studies may be required for some special cases, not always. This includes a teretogenecity study and reproductive toxicity, besides the classical parameters that are already studied.
Herb-drug interaction is another aspect less worked on, but requires a lot of documentation. Here we need help from the pharmacovigilance centers, because the adverse reaction reports are given there, so we have to improve the quality and quantity of the adverse reaction reports due to herbal products. Some training is needed and people are to be made more aware of pharmacovigilance. Then, the data available with the Poison Control Centers should be used for toxicity evaluation. We cannot ignore the importance of literature for reported toxicity that can be traditional as already mentioned in Ayurveda, any of the traditional text or other sources, and sharing of information is important.
Another important effort that has been ongoing is developing a mechanism to safeguard our traditional knowledge and this was the turning point which brought in a Traditional Knowledge Digital Library, which was initiated with the effort of AYUSH and National Institute of Science Communication and Information Resources (NISCAIR). This is an electronic database containing a translation of 36000 shlokas in five languages. The languages are English, French, Spanish, Japanese, and German. It has more than 10 million pages of information. And this has now been made available to patent offices. In the year 2009, it was made available to the European patent office, Indian Patent office, and German patent and trademark offices; and in December the United States Patent and Trademark Office (USPTO) has also been given access to it and this has saved many claims. Another aspect is, now we have a comprehensive traditional knowledge digital library, where a network for database expansion was launched in 2004, to cover approximately 2600 medicinal plants used in Ayurveda. The available toxicity data has been documented for most of the medicinal plants. Like for one particular plant Digitalis lanata (digoxin, digitoxin, digoxigenin), toxicity data is given for the oral route, for adults, for pediatric population, and individually for digoxin, digitoxin, and digoxigenin; with all sets of symptoms, usage, and antidotes. All this information is present in this database as a value addition, which is being constantly updated. So, a lot of toxicity-related information can be availed from it. Of late, we have started adding the 3D structure of the active constituent to the database. Citing this particular example, there are three constituents and the 3D structure is given there. Digitoxin has been the most worked out but docking these structures, studies show that digoxigenin may be a better / more potent therapeutic compound. So, apart from giving toxicity-related information, it also gives information regarding the possible efficacy.
What are the future approaches and challenges? Predictive toxicology is needed to prioritize for safety / toxicity testing. Like we have thousands of chemicals, we have a large number of medicinal plants and among the medicinal plants we have a whole lot of phytochemicals, so how do we prioritize them? Using Quantitative Structure Activity Relationship (QSAR) studies and building a consensus model, not taking information from one or two databases, but taking information from many models and then taking information from toxicity databases and data mining software, and then combining all of them, one comes to a conclusion. There can be an omics approach like genomics, transciptomics, metabolomics and what would it give us? These can give us noble biomarkers of toxicity, potential tissue or cellular location of the toxicity, and clues to the mechanism or mode of toxicity.
In future we would also be facing nanotized herbals because they may be more bioavailable, effective at lower doses. For example, any constituent may be an alkaloid which may be toxic, it can be nanotized and the dose-reduced, hence, reduced toxicity. Encapsulation or slow release forms can also be customized for reducing toxicity. These modifications would post some safety issues also, for which I am leaving these questions with you. Does nanotization affect the stability of herbals? Is the bio activity of nanotized herbal products similar to the parent herbal product? Do we require different safety parameters for nano herbals? Is the material used for nanotization natural or synthetic? So, whatever material is used for nanotization, its toxicity also needs to be evaluated. Leaving you with these questions and a futuristic approach.
I thank you all for your patient listening.
