Effects of extraction methods and solvent systems on extract yield, proximate composition and mineral profiling of Terminalia arjuna (Arjuna) dry powders and solvent extracts

D. K. Meena¹, A. K. Sahoo¹, H. Chowdhury¹, H. S. Swain¹, N. P. Sahu², B. K. Behera¹, P. P. Srivastava², B. K. Das¹

Introduction

The medicinal plants have been used for multifarious purposes starting from timber as fuel to drug development leaf, L5: Ethanol extract of arjuna leaf, L6: Methanol extract of Br6: Methanol extract of arjuna bark, Br7: Distilled water extract F7: Distilled water extract of arjuna fruit, AB: Arjuna Bark depend on their secondary metabolite which provides the protection to the plants under adverse environmental conditions [3]. The secondary metabolite mainly includes the bioactive compounds which are very much integral their associated organisms including fish. Bioactive compounds include the antioxidants, flavonoids, DNA damage protective compounds etc. [4]. DNA damage prevention activity indicates potential of herbal material to nullify the free radicals thereby providing the health befits and protection against de-oxygenerative diseases [5]. Crude extract of the herbal material are the witness of many untold story of the phytochemical and bioactive compounds such as anti-oxidant, flavonoids, tannin, saponin etc. present in it [6]. Since, long time anti-oxidant are being used in feed and beverage industries for extending the shelf life of the house hold material [7]. In addition, shelf-life of the feed of livestock and aqua-feed being enhanced using synthetic anti-oxidants which might be highly efficacious but relatively expensive and immunosuppressive and exert many side effects [8-9]. Gradually, the need of questing the alternate to these compounds is emerging on great pace as far as human health and ecosystem are concerned. The incorporation of such bioactive compounds in feed or other application involves a series of pre-treatment and processing for better palatability and flavors which may alter their pharmacological and ethno-medicinal properties [10]. The use of medicinal plants are increasing gradually and becoming popular remedies for common disease in day to day life [11]. The properties of medicinal plants depend on the spectrum of phytochemicals and other bioactive compounds. For instances, some plants are being recognized for their excellent ethno-medicinal properties and some are still not known till date [12]. In the era of tremendous market of medicinal plants, the developed countries are using 25% of medicinal plants out of total prescribed medicine and developing countries like India and china use 80% plants for ethno-medicinal purposes [13]. India is encompassing a diverse and better herbarium profile of excellent medicinal plants such as tulsi, neem, giloy, Terminalia arjuna etc., which have been becoming an integral part of their life and a sign of ethnicity also [14]. Terminalia arjuna (arjuna) is known for its versatility in terms of having wide spectrum of medicinal properties i.e. bark of T. arjuna is having antidysenteric, antipyretic, astringent, cardiotonic, lithotriptic, anticoagulant, hyperlipidemia, antimicrobial [15] and antiuremic properties [16]. The previous bibliographic studies on phytochemical profile of arjuna showed therapeutic and nutraceutically important compounds. Arjuna has been used in human diseases mediated through chronic degenerative actions, and showed strong antioxidant properties due to a number of important bioactive compounds. The application of Arjuna in prevention of human diseases involves either the decoction or extraction of a particular compound [17]. The success of therapeutic effects of medicinal plants depends extraction methods and solvent system and material to be extracted. Bibliographic studies showed compounds. Orbital and magnetic shaking, refluxing and recently modern techniques are being used for compound isolation etc. however, the combination either of techniques could produce better result. This is a conventional approach which apparently used without seeing the availability of bioactive compound and their fractions. Further, traditional methods are not scientifically proven and the responsible compounds for efficacy are not known. The efficacy of plant/herbal material mainly depends on its organic compounds [18]. The information on proximate composition, mineral profiling of dry powder of different parts and their solvent extraction as individual fraction and serial fraction has not been done so far. In this back drop, the present experiment was executed to evaluate the effects of processing on Arjuna dry powder of three parts and their solvents extraction on crude and serially fractionated extracts on proximate composition, yield percentage and mineral profiling.

Material and methods

Collection of the material

There parts of Arjuna plant namely; bark, leaf and fruit were selected from the trees located in ICAR-CIFRI, Campus, Kolkata, India (Authentication no. 14) and confirmed for the botanical origin with the help of morphological characters and botanist.

Preparation of the extracts

After collection, the selected parts of Arjuna were washed in tap water to get rid of the extraneous material and dirt. After washing, the plant materials were subjected to sundry till removal of the moisture. Subsequently, the material were blended in the mixer and sieved through 50 micron sieve and the course materials again were put into the mixer till the course material appears not useful. About 100 g fine powder of each part was put into the individual four different solvent in 1:5 (material: solvent) i.e. acetone, methanol, ethanol and distilled water for individual fraction and for serial fraction, it was kept in hexane to remove the greasy materials and then kept in the four solvent serially starting from one polar aprotic (acetone), and three polar protic (methanol, ethanol and o distilled water) for 36 hrs at 36 C in a shaking incubator. After 36 hrs, the respective extracts were centrifuged in ReMi R-24(ReMi, India) for 5min at 8000rpm to settle down the course particle and then supernatant was collected in separate collection vial and then supernatant was subjected to filter through Whatman No.1 (40) filter paper and the remaining residue was once again kept for overnight in their respective solvents. The same procedure was repeated once and residue was discarded for individual fraction and for serial fraction the residue was allow to dry to evaporate the mother solvent and then repeated for getting the supernatant. The supernatant was undergone the vacuum drying in rotary evaporator at below the boiling point of the solvent for proper distillation and kept till 1/10 of the original volume of the supernatant. The extracts then dried at room temperature o and kept at 4 C for further use.

Calculation of extract yield (%)

The yield (%) of solvent extracts was calculated for serial fractions and individual fractions as per the dry weight basis of the extracts with the following formula.

Weight of the extract Yield (%) = X 100 Weight of the total herbal material taken for extraction

Proximate composition

For evaluating the nutritional value, the proximate composition of the dry powder of the Arjuna extracts, individual and serial fraction were taken for analysis. The 0 samples of selected fractions were dried at 105 C to a constant weight to determine the moisture content. Crude protein was determined by measuring nitrogen (N % x 6.25) using micro Kjeldahl, crude lipid using Soxhlet apparatus and ash by combustion in a muffle furnace at 0 550 C for 6 hrs [19].

Mineral profiling

The mineral profiling was done at ICAR-National Institute of Abiotic Stress Management, MalegaonKarhavagaj Road, Khurd, Baramati, Maharashtra 413115, following AOAC [20]. Briefly, 1 g sample of each solvent extract was weighed and proceed for acidic digestion in microwave digestion system (Microwave Digestion System, Model START-D, SN- 135177, Milestone, USA). The HNO and H O were added in 5:1 3 2 2 ratio kept in digestion vessels for digestion [21]. After proper and complete digestion the samples were allowed to cool to room temperature then, digested samples were filtered with Whatman paper with 0.45 mm pore size and made up to 50 ml and proceed for trace elements analysis through Inductively coupled plasma mass spectrometry (ICP-MS) (Agilent 7700 series, Agilent Technologies, USA).

Statistical analysis

The statistical analysis was done using excel v.16, figures were edited in paint 3d v.16. The correlation matrix and box-violin plot with notches and outliers were established taking the relative value of individual fraction and serial fractions of solvent extracts in PAST 3.14 software. The values are represented as mean± standard error.

Results

Yield (%)

The yield (%) of both selected solvent extracts (individual and serial fraction) exhibits the narrow differences in yield (%) and varies from the nature of the parts used (Figure 1). Irrespective of solvents extracts the maximum yield % was recorded in the bark extracts for both the fractions, and the highest value was recorded for ethanolic extract of bark (23.6±0.026, 22.23±0.017%) followed by methanolic extract (22.7±0.016, 22.10±0.004%), acetone (11.36±0.005, 11.11±0.005%) and distilled water (4.1±0.048, 3.75±0.047%), for individual and serial fractions, respectively. The trend showing that bark extracts has maximum yield followed leaf and fruit extracts for polar protic solvents and pattern, and bark extract followed by fruit extract and leaf extracts was different in case of polar protic and polar aprotic, respectively (Table 1). The table 1 showed the comparative yield (%) of selected solvent extracts from Arjuna. The yield is a function of polarity and nature of solvents, and nature and physical structure of the herbal material to be extracted. Based on their response towards the solvent, they are assembling in clusters indicating the homogeneous groups (Figure 2). The bubble plot showing Br5 and Br6 are falling in same plain with avg. yield of more than 20%, followed by L4 and AF having yield of ≥10% and rest of the solvent extract encompassing yield ≤ 10%.

(p<0.05) with L4, L5, Br6, F6, L6, F6, Br7 and L7. Br6 has significant association with Br4, L4, Br5, L5, L6, F7

and L7.

AF 0.071±0.007 0.006±0.001 0.224±0.004 0.069±0.011 10.815±0.037 0.600±0.025 1.401±0.011 AL 0.055±0.009 0.004±.002 0.142±0.007 0.450±0.017 6.837±0.006 0.468±0.032 1.040±0.014 AB 0.051±0.010 0.002±0.154 0.115±0.006 0.441±0.017 8.198±0.005 0.373±0.040 1.036±0.014 Br4 0.052±0.010 0.006±0.069 0.166±0.006 0.486±0.015 9.653±0.004 0.437±0.034 1.802±0.008 F4 0.032±0.015 0.006±0.069 0.136±0.007 0.417±0.018 6.422±0.006 0.350±0.043 0.665±0.023 L4 0.039±0.013 0.004±0.102 0.137±0.007 0.386±0.019 8.377±0.005 0.326±0.046 0.745±0.020 Br5 0.036±0.014 0.004±0.096 0.134±0.007 0.386±0.019 7.570±0.005 0.399±0.038 0.727±0.021 F5 0.042±0.012 0.003±0.141 0.146±0.007 0.427±0.018 6.432±0.006 0.360±0.042 0.675±0.022 L5 0.042±0.012 0.003±0.120 0.176±0.006 0.480±0.016 7.982±0.005 0.411±0.037 1.422±0.011 Br6 0.058±0.009 0.004±0.099 0.155±0.006 0.473±0.016 4.816±0.008 0.471±0.032 1.774±0.008 F6 0.047±0.011 0.004±0.100 0.178±0.006 0.556±0.013 4.830±0.008 0.390±0.039 1.651±0.009 L6 0.063±0.008 0.046±0.009 0.169±0.006 0.439±0.015 5.084±0.008 0.612±0.025 1.871±0.008 Br7 0.074±0.007 0.005±0.008 0.178±0.006 0.273±0.027 7.416±0.005 0.662±0.023 1.890±0.008 F7 0.056±0.009 0.004±0.091 0.136±0.007 0.464±0.016 8.416±0.005 0.761±0.020- 1.252±0.012 L7 0.048±0.010 0.079±0.005 0.164±0.006 0.470±0.016 9.116±0.004 0.580±0.026 1.356±0.011 AF 0.076±0.065 0.076±0.002 0.226±0.005 0.697±0.017 11.238±0.002 0.600±0.025 1.387±0.001 AL 0.056±0.018 0.056±0.003 0.l153±0.069 0.454±0.025 6.899±0.003 0.468±0.032 1.027±0.001 AB 0.053±0.019 0.053±0.053 0.166±0.063 0.445±0.026 8.260±0.003 0.373±0.040 1.022±0.001 Br4 0.073±0.014 0.073±0.002 0.201±0.052 0.780±0.015 10.466±0.002 0.472±0.032 1.833±0.001 F4 0.054±0.019 0.054±0.002 0.170±0.062 0.711±0.016 7.234±0.003 0.385±0.039 0.696±0.002 L4 0.061±0.017 0.061±0.002 0.171±0.061 0.680±0.017 9.189±0.002 0.361±0.042 0.776±0.002 Br5 0.057±0.018 0.057±0.002 0.169±0.062 0.680±0.017 8.382±0.003 0.434±0.035 0.759±0.002 F5 0.064±0.016 0.064±0.003 0.180±0.058 0.721±0.016 7.244±0.003 0.395±0.038 0.706±0.002 L5 0.064±0.016 0.064±0.003 0.211±0.050 0.774±0.015 8.794±0.003 0.446±0.034 1.454±0.001 5.145±0.002 29.314±0.001 0.025±0.259 0.152±0.043 0.837±0.022 0.261±0.058 0.017±0.030 5.214±0.002 23.16±0.002 0.013±0.009 0.084±0.077 0.717±0.025 0.40±0.028 0.009±0.055 2.709±0.004 27.394±0.001 0.0141±0.046 0.090±0.072 0.707±0.025 0.158±0.095 0.009±0.055 3.148±0.003 25.114±0.002 0.016±0.040 0.112±0.058 0.775±0.023 0.169±0.089 0.015±0.033 3.508±0.003 18.967±0.002 0.0031±0.208 0.060±0.108 0.844±0.021 0.172±0.087 0.014±0.037 2.928±0.004 19.307±0.002 0.0113±0.057 0.108±0.060 0.729±0.025 0.148±0.101 0.007±0.0 68 2.940±0.004 22.138±0.002 0.0113±0.058 0.146±0.045 0.687±0.026 0.123±0.122 0.009±0.055 3.518±0.003 18.977±0.002 0.0131±0.049 0.070±0.093 0.854±0.021 0.182±0.082 0.024±0.021 4.463±0.002 26.411±0.002 0.0131±0.050 0.084±0.077 0.972±0.019 0.186±0.081 0.011±0.046 5.886±0.002 28.523±0.001 0.0159±0.041 0.170±0.038 1.401±0.017 0.033±0.457 0.033±0.015 4.868±0.002 45.816±0.001 0.0185±0.035 0.149±0.044 0.874±0.021 0.029±0.523 0.018±0.028 4.146±0.003 12.106±0.003 0.0333±0.020 0.130±0.050 0.774±0.023 0.033±0.455 0.014±0.036 4.846±0.002 17.417±0.002 0.0185±0.035 0.111±0.059 0.667±0.027 0.031±`0.478 0.018±0.028 3.885±0.003 19.528±0.002 0.0203±0.032 0.143±0.045 0.860±0.021 0.038±0.393 0.013±0.039 4.502±0.002 20.493±0.002 0.0185±0.035 0.130±0.050 0.775±0.023 0.048±0.315 0.013±0.039 5.176±0.004 29.233±0.007 0.019±0.006 0.151±0.040 0.835±0.019 0.262±0.063 0.0164±0.006 5.245±0.004 23.161±0.002 0.013±0.008 0.083±0.072 0.715±0.022 0.541±0.030 0.0087±0.011 2.740±0.007 21.399±0.002 0.014±0.008 0.089±0.067 0.705±0.023 0.160±0.103 0.0087±0.011 3.207±0.006 26.319±0.001 0.019±0.006 0.134±0.045 0.785±0.020 0.197±0.084 0.019±0.005 3.567±0.006 20.172±0.002 0.006±0.019 0.083±0.073 0.854±0.019 0.200±0.082 0.0171±0.006 2.987±0.007 20.512±0.002 0.014±0.008 0.130±0.046 0.739±0.022 0.177±0.093 0.0109±0.009 2.998±0.007 23.343±0.001 0.014±0.008 0.168±0.036 0.697±0.023 0.152±0.109 0.013±0.008 3.577±0.006 20.182±0.002 0.016±0.007 0.093±0.065 0.864±0.019 0.210±0.078 0.027±0.004 4.521±0.004 27.616±0.001 0.016±0.007 0.106±0.056 0.982±0.016 0.214±0.077 0.0145±0.007

Discussion

The multifarious role of T. arjuna cannot be overlooked particularly for ethno-medicinal properties. The mechanism behind antimicrobial properties might be due to presence of polyphenolic compounds, and the nutritional and nutraceutical value depends on mineral profiling and biochemical composition concomitant relative yield % and ash content of the compound also. In present experiment the maximum yield for both fractions namely, individual and serial was ascertained in ethanolic bark extracts but did not differ significantly (p≤0.05) from the methanolic extract of bark as individual solvent and in bark solvent extracts irrespective of solvent systems which is in accordance to Akhter et al. [22] who has reported that maximum yield was obtained from ethanolic extract of T. arjuna bark. In contrary, to the previous studies [23], where it was pointed out that generally higher extract yields, phenolic contents and plant material antioxidant activity were obtained using aqueous organic solvents, as compared to the respective absolute organic solvents. Further, Ramesh et al. [24] revealed that methanolic bark extract showed maximum yield (%) than the other solvents and parts of the T. arjuna. In present study the method of extraction was modified from orbital shaking and attempted to receive maximum bioactive principles without affecting the nature and efficacy of the compound and to obtain maximum yield which is corollary to the previous studies [23], where it was revealed that solvent extraction is most frequently used technique for isolation of plant antioxidant compounds. However, the extract yields and resulting antioxidant activities of the plant materials are strongly dependent on the nature of extracting solvent, due to the presence of different antioxidant compounds of varied chemical characteristics and polarities that may or may not be soluble in a particular solvent. In our study the combination of shaking and refluxing was used which was claimed earlier as one of the best extraction techniques for exerting the yield in terms of antioxidant principles and maximizing the yield (%) [23]. In contrast to this some researchers [25] revealed that as far as techniques are concerned, the extracts obtained by the application of sonication demonstrated significant (p≤0.05) extraction yield and bioactive compounds. The yield (%) and extraction efficiency depend on the nature and polarity of the solvent and material to be extracted. The previous studies conducted in different plant also showed the potency of the ethanol alone or in combination with other solvent systems for maximize the extraction efficiency [26-27]. From Table 1 & 2, a diverse pattern was found in proximate composition particularly for bark which has minimum moisture content and highest ash content in dry powder form in accordance to Amalraj and Gopi [14], who has reported that bark moisture and less ash content on encountering the different extraction methods and selective solvents which ascribed the inverse relationship between moisture and ash content of the selected solvent extracts might be attributed to adsorptive nature of ethanol as compared to other solvent systems and subsequently, resulted in lowering the ash content of the ethanolic bark extract. This finding is in partial agreement with Ajazuddin [28], who highlighted in his study that acid-insoluble ash value of the prepared formulation shows that a very small amount of the inorganic component is insoluble in acid. It indicates that adulteration of raw ingredients by substances, such as silica and rice husk, is very less, and a low acid-insoluble ash value may also affect the amount of the component absorbed. Ash content and mineral has linear relationship with fruit extracts and noticeably the methanolic fruit extract showed a surprising trend towards the Zn content which was reported to be as 29 mg/L in dry powder forms and could reach up to 45 mg/L in fractionated forms. Ash and moisture content showed inverse relationship which indicated that fruit extract after extraction had acid soluble ash content more as compared to other solvent extracts which is in accordance to the previous study [29], where it was pointed out that subsequent loss on drying (3.28%), ash content (1.07%), acid insoluble ash (0.26%) were observed in T. Arjuna bark extracts. In present study, for both fractions the values of yield, biochemical composition except moisture and ash and mineral profiling did not have much wider variation for a particular solvent extract, however, among the solvent extracts, a diverse patter of variation was observed. For instances, there was no relationship between ash and yield % which indicates that ash may be including acid soluble and insoluble content but yield may not deviate with the content of the ash as depicted by the fractions. In this case the maximum yield may be attributed to bioactive compounds such as polyphenols, anti-oxidants etc., which depends on type of extraction solvent and its polarity may have a significant impact on the level of extracted bioactive compounds. The polarities of the bioactive principles range from polar to non-polar, optimum extraction of polyphenols is usually obtained in the polar solvent which have a better efficiency of solvation as a result of interactions (hydrogen bonds) between the polar sites of the antioxidant compounds and the solvent than nonpolar one [30-31]. The mineral profiling also showed same trend for both fractions and particular solvent extract, as mineral profiling of plant material depend on many factors such as nature of tissue extracted, stage of plant used and parts of plant material used for extraction which is corollary to Butkute et al. [32], who summarized that mineral concentration of herbal extracts widely depending on the plant growth stage and morphological fraction, and pointed that mineral profile of young plants elements tested than the flowering one. In the present investigation it revealed that over all mineral profiling was better in terms of methanolic fruit extract which has significant ( p≤0.05) difference with the ethanolic bark extract but no significant (p>0.05) differences with the ethanolic leaf extract, ethanolic fruit extract, acetone fruit extract, and distilled water fruit extracts ,and similarly, acetone although less polar solvent however, its bark extracts has better mineral content as compared to the fruit and leaf extracts and has same pattern of significance as like as other polar solvents which ascribed to type of solvent and nature of material extracted and alteration in bonding pattern of polar aprotic solvents such as acetone which is in harmony to previous studies conducted on other medicinal plants [33-35]. From perusal of Figure 3 the solvent system are assembling in cluster as per the yield (%) concomitant increased polarity and nature of herbal material which ascribed the specific interaction and bonding between solvent and herbal material and dipolar electric point adjustment during solvation which is in accordance to Singh et al. [36]. The present study is the first of its kind which is revealing the effective solvent extract with reference to higher mineral profiling and biochemical composition and yield (%) for exploring the possibilities of their inclusion in animal and livestock feed formulation as feed ingredients or as nutraceutical.

Conclusion

From present study some of the novel and innovative results can be highlighted as follows: Solvent extraction with combination of refluxing and orbital shaking could produce better yield ascribing the potency and strength of polar solvents for extracting the biochemical principles in efficient manner. It can be recommended that bark powder can be utilized as supplement for formulating the medicated feed for livestock and fish and methanolic fruit extract to mitigate Zn deficiency and nervous disorders. Although, the mineral profiling was better in methanolic fruit extract, but not-significantly (p≥0.05 differ from ethanolic fraction, however, the ethanol can be the solvent of choice as far as the toxicity of the solvent is concerned.

Conflicts of interest

The authors declare no conflicts of interest and its original work neither communicate for publication nor under consideration for publication. Further, all authors have contributed to the work immensely.

Acknowledgement

Authors thank ICAR-CIFRI for providing the financial assistance to carry out the study. Further, the assistance from laboratory staff also thanks worthy for rendering the support during study

References

References

1. Kala, C. P.; Dhyani, P. P. and Sajwan, B. S. Developing the medicinal plants sector in Northern India: challenges and opportunities. Journal of Ethnobiology and Ethnomedicine 2006; 2: 1–15.
2. Jamshidi-Kia, L. F. and Amini-Khoei, H. Medicinal plants: Past history and future perspective. Journal of Herbal Medicine and Pharmacology 2018; 7(1): 1-7.
3. Guerriero, G.; Berni, R.; Muñoz-Sanchez, J. A.; Apone, F.; Abdel-Salam, E. M.; Qahtan, A. A.; Alatar, A. A.; Cantini, C.; Cai, G. and Hausman, J. F.; Siddiqui, K. S.; Hernandez-Sotomayor, S. M. T. and Faisal, M. Production of Plant Secondary Metabolites: Examples, Tips and Suggestions for Biotechnologists. Genes 2018; 9(6): PMC6027220.
4. Salar, R. K.; Purewal, S. R. and Sandhu, K. S. Bioactive profile, freeradical scavenging potential, DNA damage protection activity, and mycochemicals in Aspergillus awamori (MTCC 548) extracts: a novel report on filamentous fungi. 3 Biotech 2017; 7(3): 164.
5. Kalim, M. D.; Bhattacharyya, D.; Banerjee, A. and Chattopadhyay, S. Oxidative DNA damage preventive activity and antioxidant potential of plants used in Unani system of medicine. BMC Complementary and Alternative Medicine 2010; 10: 77.
6. Shad, A. A.; Ahmad, S.; Ullah, R.; AbdEl-Salam, N. M.; Fouad, H.; Ur Rehman, N.; Hussain, H. and Saeed, W. Phytochemical and biological activities of four wild medicinal plants. Scientific World Journal 2014; 2014: 857363.
7. Garcia-Alonso, F. J.; Bravo, S.; Casas, J.; Perez-Conesa, D.; Jacob, K. and Periago, M. J. Changes in antioxidant compounds during the shelf life of commercial tomato juices in different packaging materials. Journal of Agricultural and Food Chemistry 2009; 57: 6815–6822.
8. Salami, S. A.; Guinguina, A.; Agboola, J. O.; Omede, A. A.; Agbonlahor, E. M. and Tayyab, U. Review: In vivo and postmortem effects of feed antioxidants in livestock: a review of the implications on authorization of antioxidant feed additives. Animal 2016; 10: 1375–90.
9. Tiwari, R.; Latheef, S. K.; Ahmed, I.; Iqbal, H. M. N.; Bule, M. H.; Dhama, K.; Samad, H. A.; Karthik, K.; Alagawany, M.; El-Hack, M. E. A.; Yatoo, M. I. and Farag, M. R. Herbal immunomodulators, a remedial panacea for the designing and developing effective drugs and medicines: current scenario and future prospects. Current Drug Metabolism 2018; 19: 264–301.
10. Yang, C.; Kabir Chowdhurry, M. A.; Hou, Y. and Gong, J. Phytogenic compounds as alternatives to in-feed antibiotics: potentials and challenges in application. Pathogens 2015; 4: 137–56.
11. Yuan, H.; Ma, Q.; Ye, L. and Piao, G. The traditional medicine and modern medicine from natural products. Molecules 2016; 21(5): PMC6273146.
12. Atanasov, A. G.; Waltenberger, B.; Pferschy-Wenzig, E. M.; Linder, T.; Wawrosch, C.; Uhrin, P.; Temml, V.; Wang, L.; Schwaiger, S.; Heiss, E. H.; Rollinger, J. M.; Schuster, D.; Breuss, J. M.; Bochkov, V.; Mihovilovic, M. D.; Kopp, B.; Bauer, R.; Dirsch, V. M. and Stuppner, H. Discovery and resupply of pharmacologically active plant-derived natural products: A review: Biotechnogical. Advances 2015; 33: 1582– 1614.
13. Balunas, M. J. and Kinghorn, A. D. Drug discovery from medicinal plants. Life Science 2005; 78: 431–41.
14. Amalraj, A. and Gopi, S. Medicinal properties of Terminalia arjuna (Roxb.) Wight & Arn.: a review. Journal of Traditional and Complementary Medicine 2017; 7: 65–78.
15. Mandal, A.; Das, K. and Nandi, D. K. In vitro bioactivity study of bark extract of Terminalia arjuna on probiotics, commercially available probiotic formulation. International Journal of Phytopharmacology 2010; 1(2): 109–113.
16. Das, K.; Chakraborty, P. P.; Ghosh. D. and Nandi, D. K. Protective effect of aqueous extract of Terminalia arjuna against dehydrating induced oxidative stress and uremia in male rat. Iran Journal of Pharmacy Research 2010; 9(2): 153–161.
17. Meghwani, H.; Prabhakar, P.; Mohammed, S. A.; Seth, S.; Hote, M. P.; Banerjee, S. K.; Arava, S.; Ray, R. and Maulik, S. K. Beneficial effects of aqueous extract of stem bark of Terminalia arjuna (Roxb.). An ayurvedic drug in experimental pulmonary hypertension. Journal of Ethnopharmacology 2017; 197: 184–194.
18. Chikezie, P. C.; Chiedozie, O. I. and Mbagwu F. N. Bioactive principles from medicinal plants. Research Journal of Phytochemistry 2015; 9(3): 88-
20. AOAC. Official Methods of Analysis of Association of Official Analytical th Chemists, 16 Ed., 1995; 1094, AOAC Press, Arlington, VA, USA.
21. AOAC. Official Methods of Analysis of Association of Official Analytical Chemists International. In: Horwitz, W. (Ed.), 18th Ed., 2006; AOAC Press, Arlington, VA, USA.
22. Kumar, N.; Krishnani, K. K.; Meena, K. K.; Gupta. S. K. and Singh, N. P. Oxidative and cellular metabolic stress of Oreochromis mossambicus as biomarkers indicators of trace element contaminants. Chemosphere 2017; 171: 265–274.
23. Akhter, S.; Hossain, M. I.; Haque, M. A.; Shahriar M. and Bhuiyan, M. A. Phytochemical Screening, Antibacterial, Antioxidant and Cytotoxic Activity of the Bark Extract of Terminalia Arjuna. European Journal of Scientific Research 2012; 86(4): 543-552.
24. Sultana, B.; Anwar, F. and Ashraf, M. Effect of Extraction Solvent/Technique on the Antioxidant Activity of Selected Medicinal Plant Extracts. Molecules 2009;14: 2167-2180.
25. Ramesh, A. S.; Christopher, J. G.; Radhika, R.; Setty, C. R. and Thankamani, V. Comparative study on the yield ratio and bioactive J. G.; compounds of Terminalia arjuna bark and core-wood. Journal of Pharmacy Research 2010; 3(6): 1420-1422.
26. Ashraf, A.; Bhatti, I. A.; Sultana, B. and Jamil, A. Study of Variations in the Extraction Yield, Phenolic Contents and Antioxidant Activities of the Bark of F. religiosa as a Function of Extraction Procedure. Journal of Basic & Applied Sciences 2016; 12: 8-13.
27. Chatha, S. A. S.; Anwar, F.; Manzoor, M. and Bajwa, J. R. Evaluation of the antioxidant activity of rice bran extracts using different antioxidant assays. Grasas Aceites Sevilla 2006; 57: 328-335.
28. Anwar, F.; Jamil, A.; Iqbal, S. and Sheikh, M. A. Antioxidant activity of various plant extracts under ambient and accelerated storage of sunflower oil. Grasas Aceites Sevilla 2006; 57: 189-197.
29. Ajazuddin, S. S. Evaluation of physicochemical and phytochemical properties of Safoof-E-Sana, a Unani polyherbal formulation. Pharmacognosy Research 2010; 2: 318–322.
30. Verma, S. C. Quality assessment of Terminalia arjuna (roxb. ex dc.) stem bark used as cardio-protective drug and its scientific identification by hptlc using arjunic acid as marker. International Journal of Universal Pharmacy and Bio Sciences 2013; 2(6): 352-363.
31. Liu, X.; Dong, M.; Chen, X.; Jiang, M.; Lv, X. and Yan, G. Antioxidant activity and phenolics of an endophytic Xylaria sp. From Ginkgo biloba. Food Chemistry 2007; 105(2): 548–554.
32. Thouri, A.; Chahdoura, H.; El Arem, A.; Hichri, O. A.; Hassin, R. and Achour, L. Effect of solvents extraction on phytochemical components and biological activities of Tunisian date seeds (var. Korkobbi and Arechti). BMC Complementary and Alternative Medicine 2017; 17: 248–258.
33. Butkute, B.; Dagilyte, A.; Benetis, R.; Padarauskas, A.; Ceseviciene, J.; Olsauskaite, V. and Lemeziene, N. Mineral and phytochemical profiles and antioxidant activity of herbal material from two temperate Astragalus species. BioMed Research International 2018; 2018: 6318630.
34. Kazmi, N.; Hassan, W.; Hussain, S.; Khan, K.; Amir, S.; Rehman, U. R. and Riaz, A. Estimation of phytochemicals, inorganic profile and antimicrobial activity of Taxus baccata shoots. Journal of Pure and Applied Microbiology 2015; 9(1): 375-382.
35. Felhi, S.; Daoud, A.; Hajlaoui, H.; Mnafgui, K.; Gharsallah, N. and Kadri, A. Solvent extraction effects on phytochemical constituents profiles, antioxidant and antimicrobial activities and functional group analysis of Ecballium elaterium seeds and peels fruits. Food Science and Technology 2017; 37(3): 483-492.
36. Ngo, T. V.; Scarlett, C. J.; Bowyer, M. C.; Ngo, P. D. and Vuong, Q. V. Impact of different extraction solvents on bioactive compounds and antioxidant capacity from the root of Salacia chinensis L. Journal of Food Quality 2017; 2017: 9305047.
37. Singh, R. K.; Sarker, B. C; Kumbhar, B. K.; Agrawal, Y. C. and Kulshreshtha, M. K. Response surface analysis of enzyme-assisted oil extraction factors for sesame, groundnut, and sunflower seeds. Journal of Food Science and Technology 1999; 36: 511–514.