ANALYSIS OF TRACE ELEMENTS AND ORGANIC CONSTITUENTS IN MEDICINAL HERBS

Abstract

Health is a state of physical and mental well being and not merely the absence of a disease. This definition particularly fits well with the concept of Ayurveda, based on holistic approach whereas conventional medicines treat the affected part of the body only. Use of medicinal herbs in various civilizations is as old as the mankind itself. WHO estimated herbalism to be the most commonly practiced in all parts of world? In recent years, a global trend is noticed for the revival of interest in the traditional system of medicine. Screening of medicinal herbs has become a potential source of biodynamic compounds of therapeutic value in phytochemical research but little is known on the role of essential trace elements, which play a vital role in health and enzymatic processes. Popularity of medicinal herbs has also brought concerns and fears over the professionalism, quality, efficacy and safety of herbal products available in the market. Imposing regulatory standards using good agricultural, laboratory, supply and manufacturing practices only can ascertain the public belief that herbal and natural products are safe. Therefore, an extensive investigation of trace element analysis (TEA) and organic constituents are essential. Instrumental neutron activation analysis (INAA) has been used as multielemental technique for the determination of 23-31 elements in a variety of herbs and herbal formulations. AAS was used for the determination of toxic elements Ni, Cd and Pb especially. Also, thin layer (TLC) and column chromatography including preparative TLC were used for the separation of organic constituents, which were identified by ir, NMR spectral and GC-MS methods. Thesis is divided into six chapters, a brief discussion of each follows: Ch I introduces the ancient Indian medicine system using herbs and their therapeutic or medicinal uses. An overview of traditional medicines and their scientific literature is discussed. General aspects of radioanalytical methods including NAA, its different types and applications in various fields for trace element analysis (TEA) in complex biological samples are emphasized. Ageneral survey of drugs from natural sources is discussed. Literature reports on organic constituents and biocompatible trace elements have been reviewed. Lastly, Aim and Scope of the present work is described. Ch II describes Experimental Methodology and Instrumentation along with sampling methods and sample preparation in NAA and AAS. Major emphasis has been on short (iv) irradiation using pneumatic carrier facility (PCF) in DHRUVA reactor at the BARC, Mumbai resulting in the determination of 20 elements. Also included are details of highresolution y spectrometry including associated hardware and software. Modified version of phosphorus determination is described. Also our results on the participation in Intercomparison studies of Corn flour (CF-3) and Soybean flour (SBF-4) are discussed. Separation of organic compounds from the natural products is described along with a brief description of spectral identification and GC-MS methods. Ch III deals with our results on the analysis of 30 elements in 10 samples of mint {Mentha Spicata) leaves collected from four different locations in North-West India. It is enriched in Ca, Mg, K, P, Na and Fe. Variation in elemental contents from different locations is attributed to difference in soil characteristics and environmental factors. Toxic heavy metals Hg (97-983 ng/g), Sb (1.8-315 ng/g), Cd (15-722 ng/g) and As (98- 320 ng/g) are all found at sub-ppm level and vary in a wide range. Strong inverse relationship is observed between Na and Mg with CI (r= -0.95 and -0.97 respectively). An inverse correlation (r = -0.91) was observed between Zn and Cr, essential in enzymatic processes. K/Na in four different locations varies by a factor of 3 while K/P varies in a range of 2-10 with mint leaves from Dehradun showing the lowest ratio. Column and preparative TL chromatography (CHCI3/MeOH/CH3COOH in 9:2:0.5 v/v) were used to separate menthol and 1,3-dihdrocarveol in methanolic extract. Structure was elucidated by elemental analysis, ir, NMR and GC-MS studies. DPPH radical scavenging activity of diethyl ether extract was found maximum at ~40 (.ig/mL and attributed to polyhydroxy compounds. Ten hitherto unknown compounds; 2-(1-methyethylidene) cyclohexanone; 2-hydroxy 3-ethyl 2-cyclopenten-1- one; 4-ethyl 1,3-benzenediol; 4-acetyl 1-methyl cyclohexene; 2-propyl 5-methoxy phenol, carvone, octahydro-1, 4,9,9-tetramethyl methanoazulene; 2-chloro 1-ethyl 5- methoxy 3-methyl benzene; dibutyl phthalate and mono (2-ethyl hexyl ester) hexanedioic acid were identified in diethyl ether extract by GC-MS. Ch IV: After coming across news reports on Curry leaves {Murraya Koenigii) being antidiabetic and anticancerous, we analyzed 28 samples from all over India for 24 elements. Most elements vary in a wide range depending on the origin of their location. It is observed that Br, Cs, Sc, Th and Zn vary by an order of magnitude whereas Fe, Mn, Na, K, Rb, Se and P vary by a factor of 3 to 5 only. Leaves from the southern zone are enriched in K, Mg, Mn, CI and P but those from the western zone are rich in Na and (v) Zn. Concentrations of most elements from eastern zone are at par with the mean values. It is known that Cr, Fe, Cu, and Zn play an important role in the maintenance of normoglycemia by activating the p-cells of pancreas. Curry leaves are a rich source of nutrient trace elements such as Fe, Cu and Zn besides Mn, Se and minor constituents (K, Mg, Ca and P). Rb and Cs are linearly correlated (r=0.93) as their salts enhance the absorption of insulin in lower respiratory tract by lowering the breakdown of glucose. Three new compounds were separated from the ethanolic extract by GC-MS: 3- methylthiopropanenitrile (I); 1,2-benzenedicarboxylic acid, mono (2-ethylhexyl ester) (II) and 1-penten-3-ol (III). I is a plant product of biosynthesis of glucatropaelin, II an allelopathic compound reducing the need for weed management and III a strong antioxidant which can account for the antidiabetic and anticancerous properties. Ch Vdescribes the analysis of leaves (5), roots (4), fruits (3) and seeds (3) of sixteen anti diabetic herbs including three capsules (Himalaya) and five commercial herbal preparations for 6 minor and 22 trace elements. K(3.20-60.9 mg/g), Ca (4.98-47.8), Mg (0.43-1.92 mg/g), CI (0.21-11.9 mg/g) and P (0.59-6.11 mg/g) form minor constituents. All these are electrolytic or structural elements and play a role in the fluid balance. Na (0.03-5.67 mg/g) and Fe (0.11-0.27 mg/g) are found in <0.5% amounts. No single plant part is enriched in all the elements. Tejpatta, a leaf used as spice is enriched in Ca (47.8±3.5 mg/g) while roots like Kutki and Naagarmotha are enriched in Cr (2.15±0.02 ng/g) and Se (1.04±0.07 ng/g) respectively. Marodphali a fruit contains elevated concentrations of Fe, Cu and Zn, all correlated with diabetes. Seeds of Jaamun and Kaalijeera are considerably enriched in V (2.97±0.18 ug/g) and Mn (356+10 ug/g) respectively. V and Mn play an important role in controlling diabetes. Bitter gourd capsule (Himalaya) and powder (Vyas) contain very high amounts of As (1.01+0.07 ng/g and 1.44+0.12 ng/g) and Br (433+27 and 203±6 ug/g) respectively. Hg content also varies in a wide range of 23-143 ng/g but well below the permissible limit (3 ug/g). K/P were found in a range of 6.45-10.7 with a mean of 8.19+1.39. Cu and Zn are well correlated with r- 0.89. Again Rb and Cs are linearly correlated with r= 0.87. In addition five antidiabetic formulations Madhunashini (Gurukul and Divya), Diabetex (Jagdamba), Jambrushila (UAP) and Diabeticin (BACPO) were analyzed where Cu, P, Fe and Mn contents were 2-3 times higher than raw herbs and capsules while Mg content was higher by a factor of 6. Herbs and capsules have higher concentrations of Na, Ca, CI, Vand Zn. Most elements (K, Cr, Zn, Ba, Rb and Se) are in (vi) comparable range. However, toxic elements (As and Hg) are significantly lower in formulations. Rb and Cs show even better correlation with r= 0.93. Zn and Cr show an inverse correlation with r= -0.81 depicting antagonistic behaviour. Petroleum ether extracts of A. indica (neem) showed four compounds by GC-MS: 1,1,2,3-tetramethylcyclopropane, methyl phenyl sulfone, n-hexanedecanoic acid (palmitic acid) and 9,12,15-octadecatrienal. Octadecatrienal, commonly known as linolenic aldehyde, is reported in the hexane extract of kewda, an aromatic plant. Two compounds 7-(but-3-enyl) 1,2-dihydro cyclobutabenzene and 2-hydroxy methyl 1- methoxy 9,10 anthracenedione were separated from the ethyl acetate soluble fraction of petroleum extract and identified by GC-MS. Anthracenediones are a class of anti-cancer agents. Ch VI deals with the analysis of trikatu - used as a stimulant and for treatment of cold is a mixture of three spices of dried ginger (Z. Officianalis), black pepper (P. Nigrum) and pipali (P. Longum). Its five brands from Yogi (Haridwar), Vyas (Indore), Zandu (Mumbai) and Sushrut (Nagpur) Pharmacies and a local sample from Mumbai were analyzed for seven minor (Al, Ca, K, Na, P, Mg and CI) and 24 trace (As, Au, Ba, Br, Ce, Co, Cr, Cs, Cu, Eu, Fe, Hf, Hg, La, Mn, Rb, Sb, Sc, Se, Sm, Sr, Th, V and Zn) elements. It exhibits higher amounts of Ca (3.83+0.8 mg/g), Fe (0.48+0.20 mg/g), Mn (167+22 (ag/g) and Se (0.12+0.4 ug/g), which are all of nutritional importance. Some toxic heavy metals such as Sb, Hg, Th were found below permissible limits. Cu/Zn varies linearly with Zn (r = 0.92) whereas Fe and Mn exhibit inverse relationship (r = - 0.89). Ginger is particularly enriched in Ca (12.6+0.1 mg/g), Mg (1.88+0.06 mg/g), Fe (427+34 ug/g) and Mn (266+45 ug/g) whereas black pepper is enriched in P (3.53+0.35 mg/g), Cr (8.65+1.48 ug/g), Se (0.093+0.02 ug/g) and Zn (37.1+6.7 ug/g) contents. Barbituric and tannic acids were separated from the methanolic extract of pipali and confirmed by elemental analysis, ir spectra and GC-MS. The essential oil obtained by hydro distillation showed 10 compounds; 2,2-dimethyl propanoic acid, decane, 1-decyne, 3,4, 8-trimethyl 1-nonene, undecane, bis- (1-methylpropyl) disulfide, 2-nonynoic acid, 2,4-decadienal, nonanoic acid and tetradecanoic acid, by GC-MS.