Terminalia arjuna is well known for cardiotonic potential. In the present investigation different bark samples (Apical bark, middle bark and mature inner bark) of T. arjuna were subjected to inorganic analysis. The levels of Nitrogen (N), Phosphorus (P), Magnesium (Mg), Copper (Cu), Zinc (Zn), Manganese (Mn) and Iron (Fe) was higher in the apical stem bark than the middle and mature inner bark. The levels of Calcium (Ca) and heavy elements Chromium (Cr), Nickel (Ni) and Cadmium (Cd) were higher in the mature inner bark than the middle and apical bark. The Lead (Pb) was not diagnosed from all the bark samples screened.
Terminalia arjuna, one of the member of family Combretaceae, is being in use as cardioprotective drug since ancient times in Indian system of medicine. Ethno-botanically the bark is used to cure headache and to kill worms in the teeth1 and in the treatment of fever and high blood pressure2. The fresh bark juice is used as antacid3 and in the treatment of the chest pain4. Bark decoction can be used as ulcer wash while bark ash can be used in the treatment of the snakebite and scorpion sting5. The main part used as drug is crude bark of this plant. Along with most of the phytochemicals, some mineral elements play crucial role in the body defense mechanism against certain harmful diseases and few are responsible for disease development. Considering this fact and wide use of Arjuna bark as crude drug, the drug has been analyzed and standardized inorganically from pharmacological point of view.
MATERIALS AND METHODS
Total nitrogen content
Total nitrogen from the bark was estimated according to the method described by Hawk et al.6. Plant material, 0.5g oven dried powdered bark was taken in Kjeldahl’s flask. To this a pinch of microsalt(200g K2SO4 + 5g CuSO4 dehydrated) and 5ml H2SO4 (1:1) was added. To avoid bumping few glass beads were added to the flasks and the material was digested on low flame. Faint yellow coloured solution obtained after complete digestion cooled to room temperature and diluted to 100ml with distilled water. The solution was filtered through Whatman No. 1 filter paper and used for estimation of total nitrogen.
2ml plant extract was taken in a set of Nessler’s tubes. To each this tube, one drop of 8% KHSO4 was added and volume was adjusted to 35ml with distilled water. 15ml of freshly prepared Nessler’s reagent (Reagent A: 7g KI + 10g HgI2 in 40ml distilled water, Reagent B: 10 NaOH in 50ml water. ‘A’ and ‘B’ were mixed in proportion of 4:5 only at the time of estimation) was added to each test tube. The reaction between sample and the reagent gave orange brown coloured product of NH4 Hg2 I3. The intensity of this colour was measured at 520nm on a double beam UV- spectrophotometer (Shimadtzu UV-190) after 15 minutes. Total nitrogen was calculated by using standard curve obtained by using different concentrations of standard ammonium sulfate solution and employing the similar procedure as described for the analysis of samples.
Preparation of acid digests
Acid digestion method developed by Toth et al.7 was followed for the analysis of inorganic constituents. 0.5g oven dried bark powder was transferred to 100ml capacity beaker and 20ml concentrated HNO3 was added to it. The beaker was kept covered with watch glass till the primary reactions completed. The beaker was heated gently on hot plate to dissolve solid particles of bark powder. After cooling to room temperature, 60%, 10ml perchloric acid was added to it and mixed thoroughly. The beaker was again heated strongly until a clear 2-3ml colorless solution was obtained. The beaker was cooled and contents in the beaker were diluted to 100ml with distilled water and kept overnight. On the next day, extract was filtered through ashless filter paper (Whatman No.44) and filtrate was used for estimation of different inorganic constituents.
The phosphorus content was estimated according to the method developed by Sekine et al.8. Phosphorus reacts with molybdate-vanadate reagent (MV reagent) to give yellow colour complex. The intensity of yellow coloured complex was estimated by using spectrophotometer and by comparing with the colour intensity of the known standards, phosphorus content was estimated.
2ml acid digest was taken in the test tube and equal amount of 2N HNO3 was added followed by 1ml freshly prepared molybdate-vanadate reagent. Final volume in test tube was adjusted to 10ml with distilled water. The ingredients were mixed well and allowed to react for 20 minutes. After 20 minutes, colour intensity was measured at 420nm using a blank containing no phosphorus. Total phosphorus was calculated with help of standard curve obtained by using different concentrations of standard phosphorus solutions. Other steps are essentially similar as described above. Phosphorus in the plant material was expressed in g.100g-1 on dry weight basis.
The levels of Calcium, Magnesium, Iron, Manganese, Copper, Zinc, Chromium, Cadmium, Nickel and Lead were estimated using atomic absorption spectrophotometer (AAS-Model Perkins Elmer 3030). In case needed, appropriate dilutions of plant extract were made with distilled water.
RESULTS AND DISCUSSION
Quantitative estimation of different elements is shown in the Fig. 1, Fig. 2 and Fig. 3. Concentration of nitrogen was noticed higher in the apical stem bark (1.91%) during summer and during winter mature inner bark (0.17%) accumulated lowest value for total nitrogen. Total nitrogen level for middle bark was in between apical and mature inner bark and varied from 0.54% during winter to 1.22% during summer. Total nitrogen in the bark of black locust varied from 3.43% to 3.45%9. This level of total nitrogen is much higher than nitrogen values determined for Terminalia arjuna in present investigation. Though, no large differences were noticed in phosphorus content among the three bark samples during both seasons, higher level was reported in apical stem bark (0.069%) during winter than the mature inner bark (0.038%) during summer while phosphorus content was moderate in middle bark (0.045-0.052%). Davidson and Le Clerc10 estimated 0.98%, 0.95%, 0.56% and 0.70% phosphorus content in Lettuce, Spinach, Kale and Broccoli, which is much higher than phosphorus levels estimated in the present study. Calcium salts provide rigidity to the skeleton and calcium ions play a role in many, metabolic processes. Calcium deficiency give rise osteoporosis11. Level of this most immobile element was higher in the mature inner bark (2.318%) during summer and lowest value was recorded in apical stem bark (1.669%) during winter. These values are in the range of Calophyllum walkeri (2.55%)12. Magnesium is strongly electropositive, mobile and abundant divalent element in the plant and average requirement for optimal plant growth varies 0.5%-1.0%13. Magnitude of Magnesium was higher in apical stem bark during winter (0.186%) and lowest level was reported in mature inner bark (0.14%) during summer while, values for middle bark fluctuated between apical and mature inner bark. These values are higher than the Syzygium rotundifolium (0.041%)12.
Adequate copper status is essential for normal functioning of the immune system14. Copper deficiency significantly increases risk of cardiovascular disease15. In the present study amount of copper was ranged from it maximum level 2mg in apical bark during summer to lowest level 0.96mg in mature inner bark during summer. Copper content reported in Justicia adhatoda (0.838mg)16 is lower than copper reported in present study. Zinc is required for normal growth and development. Zinc is an integral component of various metalloenzymes and, along with other metals, can activate a wide variety of enzymes17. Higher level of zinc was estimated in the apical bark (2.4mg) during winter and decreased to middle bark (1.40mg) and mature inner bark (1.28) during summer. Chandrajith et al.12 determined 13.2mg zinc in the bark of Calophyllumwalkeri which is much higher than the amount of zinc reported for Terminalia arjuna in the present analysis. Manganese is distributed in tissues throughout the body and largely located in the mitochondria. Manganese deficiency disturbs the carbohydrate metabolism leading to skeletal abnormalities and impaired growth. A high concentration of Mn was observed in the apical stem bark (3.44mg) during summer and low level was reported in middle bark (0.48mg) followed by mature inner bark (0.22mg) during winter. These values are lower than the Mn content inSpinach (0.579mg) and Okra (0.382mg) reported by Audu and Lawal18. Iron, is an indispensable constituent of hemproteins like cytochrome and iron-sulphur proteins. Both in plants and animal, it plays important role in electron transport system during respiration. Iron estimated in the apical bark (25.02mg) during winter was much higher than the middle bark (13.66- 17.46mg) and mature inner bark (10.82-16.18mg). Iron content reported in the leaves of Cabbage (3.77mg) by Bosiacki and Tyksinski19 is lower than the Fe levels estimated in the bark tissue of Terminalia arjuna in the present study.
Chromium (Cr) is not naturally present in the earth crust. Almost all naturally found Cr is trivalent while, hexavalent Cr is mostly of industrial origin. Trivalent chromium (Cr3+) is most found in living organisms. Improved chromium nutrition leads to improved sugar metabolism in hypoglycemics, hyperglycemics and diabetics20. In the present study, bark samples of Terminalia arjuna contains Chromium in appreciable amount. The highest value was recorded during winter and varied from 4.12-4.88mg, while, during summer chromium content was minimum ranging from 2.32-2.2.40mg. These values are very high than Achyranthes aspera (0.148mg/100g) and Withania somnifera (0.83mg/100mg) as estimated by Jabeen et al.16. Nickel in its Ni+2 oxidation state [Ni(II)] is most common in biological systems21. Contact with nickel compounds can cause a variety of adverse effects on human health, such as dermatitis, lung fibrosis, cardiovascular and kidney diseases and cancer of the respiratory tract22. Amount of Nickel was found to be increased during winter. The highest value was recorded for mature inner bark (2.06mg/100g) and lowest for apical bark (0.04mg/100g) while in middle bark (0.06-1.98mg/100g) value was in between apical and mature inner bark. Jabeen et al.16 evaluated lower concentration of Ni in Withania somnifera (0.566mg/100g) and Justicia adhatoda (0.40mg/100g) when compared with Nickel values estimated in the present study. Cadmium is regularly found in ores together with zinc, copper and lead. Exposures to high Cd toxicity cause bone damage23and kidney damage24. Arjuna bark samples examined for cadmium content proved to contain low amount of Cd. The highest level was observed during winter than summer. The cadmium concentration increased in the order of- Apical bark (0.04-0.18mg) < Middle bark (0.1-0.2mg) < Mature inner bark (0.18-0.24mg). Kozanecka et al.25 estimated 0.05mg/100g cadmium in stem tissue of Vaccinium myrtillus and 0.140mg/100g in Convalaria maialis stem which is lower than the Cd evaluated in the present investigation. Lead was found absent in all the bark samples of Terminalia arjuna.
The bark samples of Terminalia arjuna contains substantial amount of mineral elements. The elements analyzed in the present investigation will be helpful during drug prescription and at time of drug designing. The inorganic records evaluated here will helpful to ensure the quality and verify adulteration in the natural drug in various Ayurvedic formulations.
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Fig.1. Quantitative estimation of macro elements (N, P, Ca and Mg) from Terminalia arjuna bark
Fig.2. Quantitative estimation of micro elements (Cu, Zn, Mn and Fe) from Terminalia arjuna bark
Fig.3. Quantitative estimation of heavy elements (Cr, Ni, Cd and Pb) from Terminalia arjuna bark