Phytopharmacology of Holy basil (Ocimum tenuiflorum, Ocimum sanctum) Part 3

Phytopharmacology of Holy basil
(Ocimum  tenuiflorum, Ocimum sanctum) Part 3


Phytochemistry
The chemical composition of Tulsi-Basil (Ocimum sanctum) is highly complex. The amount of biologically active compounds and nutrients may vary considerably between strains and even among plants within the same field. The effect of the way of growing, harvesting, processing and storage on the quantity of chemical constituents have not yet well understood. 
Phytochemicals found in Tulsi-Hloy Basil (Ocimum sanctum) isolated by various researchers are summarized here:
1. Eugenol, rosmarinic acid, apigenin, myretenal, luteolin, β-sitosterol, carnosic acid, flavonoids, orintin, vicenin. [50]
2. From fresh leaves and stem of Tulsi-Basil (Ocimum sanctum) phenolic compounds such as cirsilineol, circimaritin, isothymusin, apigenin, rosameric acid. The leaves of Tulsi-Basil (Ocimum sanctum) contain 0.7 % volatile oil comprising of eugenol and methyl eugenol. The oil also contains carvacrol and sesquiterpine hydrocarbon caryophyllene. Recently two flavonoids orientin and vicenin have been isolated from leaves of Tulsi-Basil (Ocimum sanctum) [51], [52]
3. The volatile oil of leaf of Tulsi-Basil (Ocimum sanctum) eugenol (as it resembles phenols it is called eugenic acid), ursolic acid, carvacrol, methyl carvicol (also called Estragol), linalool, limatrol, caryophyllene, sugars and anthocyans (anthocyanins: water-soluble pigments, that depending on their pH may appear red, purple, blue or black)
In addition phenols: rosmarinic acid, propanoic acid, apigenin, cirsimaritin, isothymucin, isothymonin and water-soluble flavonoids: orientin and vicenin have been isolated. 
The stem and leaves contain a variety of biologically active compounds of groups: phenols, organic acids, saponins, flavonoids, triterpenoids and tannins.
The volatile oil of seeds have fatty acids, sitosterol, mucilage, some sugars (xylose) and polysaccharides. [53]
4. Chemical constituents of Tulsi-Basil (Ocimum sanctum) in different solvents were found to be different. Here I summarize results of various researchers. 
a) Hexane (H): 1,2-dimethoxy-4-(2-propyl)benzene, 2-pentanone, caryophyllene oxide, acetic anhydride, tricycloundecan, aromadendrene oxide and benzodioxide 
b) Ethyl acetate (EA): 1, 2-dimethoxy-4-(2-propenyl)benzene, 2-pentanone, 4-methyl-2-pentyl acetate, aromadendrene oxide, carbophyllene oxide, benzenedicarboxylic acid and 10-heneicosene
c) Chloroform: 1, 2- dimethoxy-4-(2-propenyl) benzene, 4-methyl-2-phenyl acetate, ledene oxide, longipinocarvone, phtalic acid, butyl hexyl ester, 2-pentanone, eugenol, 1.2-benzene dicarboxylic acid and 2-4-(lpropyl)-(E)-phenol
There are many essential oil constituents in Tulsi-Basil (Ocimum sanctum) such as camphor, vanillin, naphthalene, ledene oxide, caryophyllene and eugenol. But when extracted with different solvents the extract contains only borneol, vanillin, naphthalene and eugenol. [54]
Seeds
In addition to mucilage, some sugars such as xylose and polysaccharides seeds of Tulsi-Basil (Ocimum sanctum) contain ‘drying’oil with physico-chemical properties similar to those of linseed oil. Other unsaturated fatty acids include α-linoleinic acid, linoleic acid and oleic acid. The most abundant saturated fatty acids are palmitic acid and stearic acid.
The composition of Tulsi-Basil (Ocimum sanctum) seed oil suggests that the oil would be suitable for industrial purposes, much in the same way as linseed oil is used. [55]   
Nutrients and mineral contents (Fresh leaves) Per 100 G
Protein 4.2 g, Carbohydrate 2.3 g, Fat 0.5g, Omega 3 fatty acids 3%
Vitamin A 3%, Vitamin K 13%, Vitamin C 5%, Folate 4%
Calcium 4-5%, Iron 4-5%, Manganese 1.5-3 %, Copper 9% [56]
Inorganic elements reported by other researchers are: Sodium 0.74%, Potassium 6.62%, Sulphur 1.55%, Phosphorus 1.10% and Nitrogen 3.30%
Recent advances on chemical composition of Tulsi-Holy Basil (Ocimum sanctum)
Leaves
The leaves contain high amount of phytochemicals which include: Toluene, Camphene, Octane, Benzene, Citronellal, Sabinene, Limocene, Ledol, Dimethylbenzene, ethyl-2-methylbuteryrate, Eugenol, Iso-eugenol, Terpiniolene, β-elemene, Iso-caryphyllene, α-amorphene, α-amorphene, α-guaiene, α-humulene, humulene oxide, 14-hydroxy-α-humulene, α-terpenol, borneol, Calamine, Nerolidol, Carvacrol, Geraneol, Elemol, Tetradecanol, (EZ)-famesol, Cissesquisainenehydrate, α-bisbolol, Selin-11-en-4-α-ol, α-murolene
The extraction of fresh leaves and stem yielded phenolic compounds such as Apigenin, Circimaritin, Isothymusin, Eugenol and Rosameric acid.
Tulsi-Basil (Ocimum sanctum) is also a good source of monoterpenes and sesquiterpenes like Neral, Camphene, Cholesterol and Stigmasterol along with Vitamin A, Vitamin C and Vitamin K [57]
Identity, Purity and strength as per international guidelines
Foreign matter: Not more than 2 percent
Total Ash: Not more than 10 percent
Acid-insoluble Ash: Not more than 1.5 percent
Sulfated ash: Not more than 20 percent
Alcohol-soluble extractive: Not more than 4 percent
Water soluble extractive: Not less than 8 percent
Loss on drying: Not more than 14 percent [58], [59]

Heavy Metal Analysis as per international guidelines
Element              Permissible Limits
Arsenic                 Not more than 5 to 10 mg/kg
Cadmium              Not more than 0.03mg/kg    
Lead                      Not more than 5 to 10 mg/kg                   
Mercury                Not more than 0.5 mg/kg
Chromium            Not more than 0.3 mg/kg [60], [61]

Permissible Microbial Load as per international guidelines
Microbial Limits:
Total bacterial count:                                   Not more than 105cfu/g
Total yeast and mould count:                      Not more than 104cfu/g
Bile tolerant gram negative bacteria:           Not more than 104cfu/g [62], [63]
Specific Pathogens: (as per international guidelines)
Salmonella species:                Absent in 25 g /none
Escherichia coli:                     Absent in 1g / maximum 102 to 104 per gram 
Staphylococcus aureus:         Absent in 1g          
Pseudomonas aeruginosa:     Absent in 1g
Shigella species:                     Absent in 1g                
Enterobacter species:            maximum 104 per gram 
Other enterobacteria:            maximum 103 per gram
Aerobic bacteria:                  maximum 105 to 107 per gram  
Mould propagules:               maximum 103 to 105 per gram    
Yeasts and Mould:               maximum 103 to 104 per gram [64], [65]       

Aflatoxins (as per international guidelines)

Aflatoxin B1, Aflatoxin B2, Aflatoxin G1, Aflatoxin G2                      

Preferably Aflatoxins should be below detectable limits (BLD) [66], [67]

Pesticide residues (as per international guidelines)
In recent times various pesticides are used to protect and preserve the food and medicinal values of plants.
To avoid toxicity of herbal medicine, International Society for Standardization of Drugs and World Health Organization (WHO) have laid the guidelines for permissible levels of pesticides in herbal medicines.
In general, the pesticide contamination in any herbal medicine should be less than 1 percent of total intake from all sources, including food and drinking water.
Aldrin and dieldrin are broad spectrum pesticides commonly used in agriculture. The recommended maximum limit of these pesticides is Not more than 0.05 mg/kg. [68], [69]
Radioactive residues (as per international guidelines)
A certain amount of exposure to ionizing radiation of plants cannot be avoided since there are many sources, including radionuclides occurring naturally in the ground and the atmosphere.
The World Health Organization (WHO), in close collaboration with several international organizations, has developed guidelines for permissible and acceptable limits for radioactive residues in herbal medicines.
The amount of radiation in plants depends on intake of radionuclides. Significant risk is associated only with consumption of quantities over 20 kg of plant material per year so that the risk to health is most unlikely to be encountered given the amount of medicinal plant materials need to be ingested. Additionally, the level of contamination might be reduced during the manufacturing process. Therefore World Health Organization (WHO) has not proposed strict limits regarding the acceptability for radioactive contamination. [70]
Genetic Identity/ DNA sequencing

The genus Ocimum is highly variable. It includes countless species. Further the     genus possesses wide genetic diversity at intra-species and inter-species levels. It is very difficult to identify species on the basis of morphology of the leaf alone. The recent technique of genome sequencing has offered a helping hand in identifying each species accurately. [71]

Gene sequencing will now facilitate identification of not yet identified genes involved in the synthesis of important secondary metabolites in Tulsi-Basil (Ocimum sanctum).
According to the scientists, the development of molecular tools and genomic resources will accelerate molecular breeding and utility of Tulsi-Basil (Ocimum sanctum) for medicinal purpose. [72]
Tulsi-Basil (Ocimum sanctum) is reported to have a karyotype of 2n=36, which is the lowest among members of Ocimum genus. [73]
PCR Analysis and SSR Sequencing

Recently the expression of six important genes identified from genome sequencing data were validated by scientists by performing q-RT-PCR in different tissues of five different species, which shows the high content of urosolic acid-producing genes in young leaves of Rama Tulsi (Ocimum sanctum) subtype. In addition, the presence of eugenol and ursolic acid, implied as potential drugs in the cure of many diseases. These findings were confirmed by using mass spectrometry.
Expression of anthocyanin biosynthesis-related genes in leaf samples of Krishna Tulsi (Ocimum tenuiflorum) were found to be high explaining the purple coloration of Krishna Tulsi (Ocimum tenuiflorum).  [74]
By suing SSR method, efforts are now made by researchers towards the development of molecular markers in order to study genetic diversity of Ocimum species. The data generated from SSR study would be helpful in providing insights to the plant breeders and geneticists for evaluation of desired genotypes with varied essential oil compositions and also development of new Ocimum species. These efforts will fling open the door of goldmine of synthesis of new drugs. 
RAPD analysis
RAPD (Random Amplification of Polymorphic DNA) analysis has helped scientists to identify Ocimum species more accurately. [75]

AFLP Analysis

AFLP (Amplified Fragment Length Polymorphism) analysis also helps to identify Ocimum species accurately. The advantages of AFLP (Amplified Fragment Length Polymorphism) over other genetic markers are: high reproducibility, resolution, sensitivity at whole genome level and no need of prior sequence information for amplification. AFLP (Amplified Fragment Length Polymorphism) also has capability to amplify between 50 and 100 fragments at a time. [76]
HPTLC analysis
By HPTLC analysis quantitative estimation of various phytochemicals such as eugenol, ursolic acid, rosmarinic acid and many others in Tulsi-Basil (Ocimum sanctum) has been done.
Chromosomal Identity
Ocimum species are characterized by the different basic chromosome numbers
X=8, 10 12 or 16. In case of Ocimum tenuiflorim individuals chromosome numbers reported are 2n= 32, 2n=36 and 2n=76, however internationally accepted chromosome number of Ocimum tenuiflorim is 2n=36.
As of today internationally accepted number of chromosomes in other Ocimum species are:
Ocimum gratissimum:     2n= 40
Ocimum basilicum:         2n=48
Ocimum americanum:    2n=72
Lemon basil:                  2n=52   [77]
Ocimum canum Sims (hoary basil):        2n=26
Ocimum kilimandscharicum:                   2n=76 [78]
Ocimum viride Willd:              2n=40
Ocimum suave Willd:              2n=48 
Vij and Kashyap (1976) have counted chromosome number 2=64 for Ocimum americanum collected from North India, while Singh et al (1980) reported chromosome count of 2n=24, 26, 72 and 84 in Ocimum americanum L.
Morton (1962) has observed chromosome number 2n= 72 for Ocimum canum collected from North India.
Pushpangdan and Sobti (1982) have reported chromosome number 2n=24 for Ocimum canum introduced from Kenya and 2n=26 for Ocimum canum growing in South India. Mukherjee and Datta (2006) also reported chromosome number 2n=26 for Ocimum canum.   
Pushpangdan and Sobti (1982) have reported chromosome number 2n=48 for Ocimum basilicum growing in West India while Paton and Putievsky (1996) have counted 2n= 64, 72, 74 and 76 for Ocimum basilicum. For Ocimum basilicum var. crispum Mukherjee et al (2005) reported a chromosomal count 2n=52 and Eder and Aikpokpodion 2n=48 and 60.  [79] 

References

50. Baliga M. S. et al, Ocimum sanctum L (Holy Basil or Tulsi) and its phytochemicals in the prevention and treatment of cancer, Nutr Cancer, 2013; 65 Suppl 1:26-35
51. Uma Devi P. et al, Radioprotective, anticarcinogenic and antioxidant properties of the Indian holy basil, Ocimum sanctum (Tulsi). Indian J Exp Biol. 2001; 39:185-190
52. Gupta et al, Validation of traditional claim of Tulsi, Ocimum sanctum Linn. as a medicinal plant. Indian J Exp Biol. 2002; 5:765-773
53. Priyabrata Pattanayak et al, Ocimum sanctum Linn. A reservoir plant for therapeutic applications: An overview. Pharmacogn Rev. 2010 Jan-Jun; 4(7): 95-105
54. N. Dev et al, Chemical Compositions of different Extracts of Ocimum basilicum, Journal of Scientific Research, 3(1), 197-206 (2011)      
55. Paul Angers et al, Basil seed oils
https:// hort.purdue.edu/newcrop/proceedings1996/V3_598.html
56. Priyanka Pattanayak et al, Ocimum sanctum Linn. A reservoir plant for therapeutic applications: An overview Pharmacogn Rev 2010 Jan; 4(7): 95-105
57. Bano N et al, Pharmcological Evaluation of Ocimum sanctum, Journal of bioquivanance & Bioavailability, Volume 9(3): 387-392 (2017)
58. Quality control methods for medicinal plant materials. Geneva, World Health Organization, 1998.
60. Quality control methods for medicinal plant materials. Geneva, World Health Organization, 1998.
62. Quality control methods for medicinal plant materials. Geneva, World Health Organization, 1998.
64. Quality control methods for medicinal plant materials. Geneva, World Health Organization, 1998.
66. Quality control methods for medicinal plant materials. Geneva, World Health Organization, 1998.
68. Guidelines for predicting dietary intake of pesticide residues, 2 nd review edition, Geneva, World Health Organization, 1997
69. European pharmacopoeia, 3rd edition Strasbourg, Council of Europe, 1996 
70. Quality control methods for medicinal plant materials, Geneva, World Health Organization, 1998 
71. Atul K. Upadhyay et al, Genome sequencing of herb Tulsi (Ocimum tenuiflorum) unravels key genes behind its strong medicinal properties, BMC Plant Biol. 2015; 15:212
Scientists-sequence-tulasi-genome.html 
73. Felix Bast et al, Chloroplast DNAPhylogeography of Holy Basil (Ocimum tenuiflorum) in Indian Subcontinent, The Scientific World Journal, Volume 2014, Article ID 847482
74. Atul K. Upadhyay et al, Genome sequencing of herb Tulsi (Ocimum tenuiflorum) unravels key genes behind its strong medicinal properties, BMC Plant Biol. 2015; 15:212
75. Carovic-Stanko et al. Genetic relations among basil taxa (Ocimum L.) based on molecular markers, nuclear DNA content and chromosome number, Plant Syst. Evol. 285, 13-22. IJPPR, Volume 8, Issue 2: February 2016
76. Carovic-Stanko et al. Genetic relations among basil taxa (Ocimum L.) based on molecular markers, nuclear DNA content and chromosome number, Plant Syst. Evol. 285, 13-22. IJPPR, Volume 8, Issue 2: February 2016
77. https://www.ncbi.nlm.nih.gov>pmc>articles>PMC4552454
IJPPR, Volume 8, Issue 2: February 2016
78. Moumita Mukherjee and Animesh K. Datta, Secondary Chromosome Association in Ocimum spp., Cytologia 71(2):149-152, 2006 
79. Johnson Ademola Idowu and Matthew Oziegbe, Mitotic and Meiotic Studies on Two Species of Ocimum (Lamiaceae) and their F1 Hybrids, Botanica Lithuanica 2017, 23(1): 59-67



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