Importance of Genetic Identification of Medicinal Plants

Importance of Genetic Identification of Medicinal Plants

Bhumyamalaki       
Thyagarajan’s landmark paper published in ‘The Lancet’ on eradication of Hepatitis B surface antigen by using ‘Bhumyamalaki’ or ‘Bhuiawala’ (Phyllanthus amarus) in HBV carriers stirred the medical fraternity. Bloomberg et al added value to Thyagarajan’s paper, for their results were much better than Thyagarajan’s. Inspired by this data many researchers, clinicians and specialists in Ayurveda started using ‘Bhumyamalaki’ or ‘Bhuiaawalaa’ (then called Phyllanthus niruri) to treat jaundice in general and viral hepatitis B in particular; but in vain for they did not get the desired results.
Despite the uncertain, unconvincing and inconclusive results, the trade of ‘Bhumyamalaki’ or ‘Bhuiaawalaa’ (so called Phyllanthus niruri) reached its zenith. 
I had an opportunity to discuss this issue with a professor of pharmacology. The professor stunned me by saying ‘the results of the Chinese Phyllanthus amarus are much superior in this regard to the Indian Phyllanthus amarus. As I asked the explanation, ‘Purity of the plant’ the professor replied.
Since ancient times, herbs of the genus Phyllanthus have been in use in traditional European, Chinese and Indian medicine mainly for treating jaundice. Modern research with Phyllanthus focuses on its potential for fighting hepatitis B virus and malaria parasite.
The species Phyllanthus niruri is an American species and not at all found in India. In India Phyllanthus amarus, Phyllanthus fraternus, Phyllanthus debilis and Phyllanthus urinaria grow sympatrically (in the same geographical region or zone). Morphologically they bear a remarkable semblance to Phyllanthus niruri. (See images). Earlier researchers therefore grouped them in a single group of species Phyllanthus niruri which was later described as ‘niruri complex’. However taxonomically this is incorrect. Each species must be identified separately and individually with a specific taxonomical (botanical) name.

                                          
               Phyllanthus niruri                           Phyllanthus amarus

                                       
                         lanthus fraternus                                Phyllanthus debilis       

         
               Phyllanthus urinaria                        Phyllanthus megalanthus

                
           Phyllanthus myrtifolius                  Phyllanthus pulcher

                
              Phyllanthus talbotii                             Phyllanthus tenellus                 [1]

The confusion still exists because of morphological similarities. This implies inadvertent or deliberate adulteration of crude drugs lowering the efficiency of the medication for its intended purpose. Secondly, incorrect identification may also lead to erroneous reports on actions of the herb. These may be some of the reasons why researchers could not emulate the results reported by Thyagarajan and Bloomberg et al.
To overcome this problem in crude drug and compliment morphological identification in live plant, scientists offered genetic identity for each species. This is based on SCAR (Sequence Characterized Amplified Region) markers of DNA. The genetic identification ofPhyllanthus species mentioned above is:
Phyllanthus amarus:  1150 bp
Phyllanthus fraternus: 317 bp
Phyllanthus debilis: 980 bp
Phyllanthus urinaria: 550 bp [2], [3]

Bhringraj

In Ayurveda, Bhringraj was extolled as ‘hair tonic’ (Kesharaaja). Later it was described to be useful for the disorders of liver and spleen.According to the colors of the flowers the herb bears; in Ayurveda three varieties of Bhringraj are described: White, Yellow and Neela Bhringraj or Neeli Bhringraj.


                                                                                                                                                                                   
             Shweta Bhringraj                                        Peeta Bhringraj
               (Eclipta alba)                                         (Wedelia calendulacea)                                                                              
   
        
                                                 Neela Bhringraj (Indigofera tinctoria)     [4]

Although they have some similar Ayurvedic and pharmacological actions; taxonomically they are three different plants: 
Shweta (white) Bhringraj: Eclipta alba
Peeta (yellow) Bhringraj: Wedelia calendulacea
Neela (blue) Bhringraj: Indigofera tinctoria
(For more details see below: Confusion due to taxonomical synonyms)

Daruharidra

In field, usually more than one species of Berberis (Daruharidra) grow together (Sympatrically).  Morphologically many of them resemble each other. In 1999 Rao et al. reported pollen morphologic studies in solving taxonomic confusion. However during harvesting, in the field, it becomes difficult to distinguish one species from the other. Thus species other than Berberis aristata are also harvested. Since stems of the plant are used for medicinal purpose, it becomes even more challenging to obtain authentic collections and assure quality. Conventional Pharmacognosy techniques based on macro and micro-morphological characters of plant identification may not be effective in distinguishing Berberis aristata because some species may share similar histological characteristics, making microscopic identification inaccurate. Different species of the same genus may have totally different or weaker pharmacological action compared with the authentic species. DNA-based markers have therefore become a popular means of definitive identification of plants because genetic composition is unique for each individual species irrespective of physical form, age, physiological condition, environmental factors, storage and processing.            
The genetic identity of Berberis aristata is determined by “Direct sequencing of complete ITS region” 5
This reveals that, from traditional herbalists, Ayurvedic physicians to taxonomists it seems there exists confusion regarding ‘appropriate’ and ‘accurate’ identification of medicinal plants.
The factors producing confusion in appropriate and accurate identity of Ayurvedic plants are:
1. Confusion due to morphological similarities:
Phyllanthus niruri and Phyllanthus amarus both look alike. Hence they are confused with each other. However P. niruri is not an Indian plant but P. amarus is!
2. Confusion due to taxonomical synonyms:
The aim of Carl Linnaeus a Swedish botanist, zoologist and physician in developing a system of botanical nomenclature (taxonomy) was to avoid confusion in identification of plants (and animals) and impart one universal scientific name to each species. Because of this taxonomical system any given species, be it a plant or an animal, is appropriately and accurately identifiable world over.
 But, even today taxonomically there exist many synonyms of Bhringraj, such as Eclipta alba, Eclipta prostrata, Eclipta erecta and many more. This must not happen.    
3. Confusion due to similarities in pharmacological actions:
Shweta (white) Bhringraj, Peeta (yellow) Bhringraj, and Neela (blue) Bhringraj have very similar pharmacological actions. All the three plants have similar leaves and similar ‘morphological look’. Hence in Ayurveda three varieties of Bhringraj are described. Taxonomically however, they are three different plants; three different species: Eclipta alba, Wedelia calendulacea and Indigofera tinctoria.Again,Wedelia calendulacea and Wedelia chinensis are used as synonyms. Surprisingly, even today Wedelia calendulacea is known as Peeta (yellow) Bhringraj and is used in place of Eclipta alba. Scientifically this is adulteration!
Hence there is an absolute need for ‘genetic identification’ of Ayurvedic plants!
Currently various methods and techniques are used for genetic identification. The detailed discussion is beyond the scope of this work. Hence I prefer to mention some commonly used types of genetic markers.
RFLP (Restriction Fragment Length Polymorphism)
AFLP (Amplified Fragment Length Polymorphism)
RAPD (Random Amplification of Polymorphic DNA)
VNTR (Variable Number Tandem Repeat)
MSP (Micro Satellite Polymorphism)
SNP (Single Nucleotide Polymorphism)
STR (Short Tandem Repeat)
SPF (Single Feature Polymorphism)
 SCAR (Sequence Characterized Amplified Region) (6), (7)

References:

1. Images from Wikipedia and google images 
2. http://staff.cimap.res.in/PublicationFiles/Planta_Med_2008_74_296%E2%80%93301..pdf
3. Jain N, Shasany AK, Singh S, Khanuja SP, Kumar S; SCAR markers for correct identification of Phyllanthus amarus, P. fraternus, P. debilis and P. urinaria used in scientific investigations and dry leaf bulk herb trade; Planta Med, 2008 Feb; 74 (3): 296-301  
4. Google images
 5. Subramani Paranthaman Balasubramani, Gurinder Singh Goraya and Padma Venkata Subramanian; Development of ITS sequence-based markers to distinguish Berberis aristata DC from B. lyceumRoyale and B. asiatica Roxb.; Biotech. 2011 February 25; 1(1): 57
6. K Semagen, A. Bjornstad and M. N. Ndjiondjop, An overview of molecular marker methods for plants; American Journal of Biotechnology Vol. 5 (25) pp2540-2568 December, 2006
7. P. Kumar, V.K. Gupta, A. K. Misra, D. R. Modi and B. K. Pandey, Potential of Molecular Markers in Plant Biotechnology; Plant Omics Journal Southern Cross Journals, 2 (4):141-162 (2009)



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