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174
Ethno Pharmacological Screening of Selected Compounds from Crataegus Rhipidophylla Plants Grown at Swat, Pakistan
Sabih Ullah1, Imran Ullah1* ,Zeeshan Shahzad2*, Saif ullah3, Abdul Jawad3,Muhammad Arif3, Muhammad Fazil3, Mahir Ali3, Salman Khan2, Fazli Maula3, Ismail khan3, Ismail ahmad4
1 Department of Biochemistry Hazara University Mansehra Kp Pakistan
2 Department of Biotechnology & Genetic Engineering Hazara University Mansehra KP Pakistan.
3. Department of Botany Government Degree College lahore Swabi Kp Pakistan 4.Department of Zoology Abdul wali khan university Mardan KP Pakistan.
Corresponding Author: Imran Ullah1* Email Address: [email protected]
Co-Corresponding: Zeeshan Shahzad2*
Email Address: [email protected]
Abstract
The present study involves the photochemical screening, mineral analysis and biological activities of crategus rhipidophylaa. Crataegus rhipidophylla collected from district Swat. It is medicinal plant.
We used solvent methanol, chloroform, aqueous extract of whole plant of crategus rhipidophylla.
The whole parts of the selected medicinal plant were washed, shade dried and then powdered for qualitative analysis. Aqueous, methanol and n-hexane extracts of whole plant were used to find out the photochemical constituents, mineral analysis, antioxidant and antibacterial activity in the crataegus rhipidophylla. The results of the phytochemical analysis of crategus rhipidophylla showed that the terpenoids, tannin, flavonoids, glycosides, alkaloids, saponins were present in all extracts except of rare phytochemicals. Mineral analysis showed that the plant extract contain copper (0.063mg/L), iron (mgL62.26/), manganese (0.448mg/L), lead (0.627mg/L), zinc (0.757mgL/), cadmium (0.211mg/L), chromium (1.332mg/L), cobalt (0.419mgL/). Antibacterail activity was carried out against five different bacterial strains: Esheria coli, Pseudomonas Aeruoginosa, Salmonella typhae, Shigella, Staphylococcus aureus. The antibacterial activity was determined by agar method. Antioxidant activity was carried out by DPPH method. The highest antibacterial activity; 15±0.99 mm in Hexane extract against S. aereus and 15±0.85 mm in methanol were obtained against Samonellatyphae respectively. The highest antioxidant activity was observed at 80ppm n- hexane (86.65) second highest activity was observed at 60ppm n-hexane(71.98) methanol was present at 80ppm (71.87) it was also observed that this plant is potential source of antioxidant agent and the sample has good quantity of phytochemicals. The current investigation will help the researcher as basic information for future research in manipulating the buried potential of this important plant which has not been explored so far.
1: Introduction
All around the world medicinal plants possess great influence on the health of individuals. Medicinal values of these plants are mainly based on chemical constituents they possess and their action of physiological on human beings. These biological constituents are phenolic content, alkaloids content, flavonoids and tannin content. Medicinal plants can also be used as a food plants in all over the
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175 world. These plants are also be used as for nursing or pregnant mothers for medicinal dedications (Adewusi, E.A. and Afolayan, A.J., 2010). From many centuries ago till now medicinal plants are utilized as a major source for medication and pharmaceutics. For synergistic neutralizing reactions there are different kinds of active compounds present in medicinal plants. The essential advantages of medicinal which are obtained from plants are comparatively harmless than synthetic, have high therapeutic benefits and high cost-effective treatment (fatima., et al., 2020).
Crataegus rhipidophylla is one of the most essential medicinal plants generally found in America, Asia and Europe. In Asia including Pakistan its cultivated in the northern hilly areas of the country i.e. at high altitude regions about 1850—3000 m height. Crataegus rhipidophylla belong to Rosaceae family which are categorizes in medium sized flowering plants (Faheem et al., 2020; Faheem,et al.,2012 ).
Crataegus rhipidophylla can be found as a shrub or tree and their length range from up to 7 meters (23 ft) tall. Leaf blades are dark green, with 2-4 pairs of acute or sub acuminate lobes. Its heavy thorns can be up to 1.5 cm long with white flowers while fruits are bright or dark red, 8–15 mm long and 1.3-2 times as long as wide (Donmez and Ali, 2007). Consumption of Crataegus rhipidophylla as a medicicinal plants have a lot of therapeutic benefits including reduced the risk of cardiovascular diseases, Central nervous system abnormality, as well as act as an anti-hepatic carcinoma and anti- cancer [Kashyap CP et al., 2012; Ju LY. 2005 ]. Globally Crataegus rhipidophylla hurbs are widely used and processed as a jam, wine, candy, vinegar, and also used in cosmetic remedies [Faheem et al., 2002]. These medicinal herbs contain vital curative ingredients including important secondary metabolites, phytochemicals, carbohydrates, flavenols, phenolics compounds, terpenoids, Choline, and amines containing compounds. Crataegus rhipidophylla compounds are described to have anti- oxidative potential to diminish the harmful free radicals, inhibit chronic inflammation infections, decease anxiety, anti-diabetes, prevent obesity, liver diseases, maintaining renal failure, anti- asthmatic and anti-asthmatic activities (Kashyap CP et al., 2012). Crataegus Rhipodophylla occurs naturally in Kohistan, a district in KP province of Pakistan. There are many studied reported on crataegus but current study is designed to investigate phytochemical screening, antioxidant activities and antimicrobial analysis that are not reported yet. Hence, the recent research aims to recognize the metabolite flux, chemical composition, and different levels within tissues of C. rhipidophylla and develop the chemical signatures for quality control and future scientific investigations
2: Materials and methods 2.1: Sample Collection
Fresh and young plant sample of crataegus rhpidophyllum (shown in Fig.1) were collected in different regions of District Kohistan, KPK Pakistan. Collection of fresh and young samples of crataegus rhpidophyllum was done at the same time from all regions in order to minimized environmental variations. Collected material of plant was washed with tape water for removal of dust particles and then placed in dark room for drying purpose.
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176 Figure 1: Plant of crataegus rhpidophyllum
2.2 Processing of Dried Plants
Once the plant sample dried up they were converted to fine powder with the help of juicer, motor and pistils. Plant powders were transferred to Autoclaved tight air bags for further analysis.
2.3 Preparation of plant extract
For extract preparation of plant sample four different solvents were used. 50g of plant sample were kept in 500mL of each solvent and kept in shaking incubated for 2 weeks. After storage of plant sample in solvents, rotary evaporator was used for evaporation of solvents from the plant material. Semi solid liquid was obtain after performing rotary evaporator, placed in label beaker and again store in desiccators for removal of remaining solvents. After complete evaporation of solvents different solvents extract were collected that was used for further analysis.
2.4 Phytochemical analysis
Phytochemical screening of the subject samples for the qualitative identification of tannin, saponin, terpenoids, cardiac glycosides, alkaloids, flavonoids was done by various analytical techniques in NTHRI (National tea research center, shinkiari) Pakistan (Khan et al., 2011).
2.5 DPPH activity for determination of antioxidant potential
Activity of antioxidant of the plant materials were determined by using reported method of (Yen and Duh (1994). Stock solution of DPPH was prepared by mixing 0.015g of DPPH solution in 100mL of methanol. Working solution of DPPH was prepared by diluting with methanol until its absorbance reached to 0.98(±0.02) at 517nm on spectrophotometer. Plant sample were prepared in different dilutions (50-500μg/mL). 400μL of plant sample was mixed with 3.6mL of DPPH working solution and incubated for 30 min. After incubation absorbance of this sample was checked 517nm by using UV visible spectrophotometer. For calculation of antioxidant capacity of the plant material following formula was used:
% Antioxidant Capacity= [1 –As/Ac] x 100
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177 Where As is the sample absorbance and Ac is the control absorbance. Ascorbic acid was used as a standard.
2.6 Antimicrobial activity of the plant sample
Antimicrobial activity of the plant material was determined by using reported method (Zain et al., 2012). Agar plate was inoculated by scattering of microbial inoculum over the surface of microbes with help of sterilize cotton swab. Cork borer sterilizer was used for holes formation in each well of agar plate and sample injection was done by dropper. Specific types of antibiotics were used for validation of this activity and pasted on surface of agar well plate. After injection of sample and standard then plates were allowed to incubate for 24 hours at room temperature. Measuring scale was used to determine zone of inhibition of the sample and standard to determine.
2.7 Mineral analysis
Entirely the minerals were estimated by using the methodology of atomic absorption spectrophotometer (AAS).
3: Results and discussion
3.1: Phytochemical analysis
Comparative analysis of screening of 6 different tests was done in different fractions of crataegus rhpidophyllum of having aqueous, chloroform, hexane and methanol as shown in Table 1, Figure 2. After comparative analysis we revealed that screening test for flavonoids, terpenoids and glycosides were found positive in all four fractions of plant sample while other fractions showed variable results of present in one fraction while absent in other fraction. Negative results of the extract might be due to solubility in which respective compounds don’t dissolve. Some research data showed that the both medicinal and edible plants possess secondary metabolites which can show different pharmacological activities. A class of flavonoids known as is flavones can be used to triggered bacterial, viral and inflammatory effects (Liu et al., 2019). Previously reported data of (Alhakmani, F et al., 2013) showed that these compounds can mediate antioxidant and metal chelating activity.
Table 1: Comparative analysis of different screening tests
S.No Test Plant sample
Aqueous Chloroform Hexane Methanol
1 Saponins + - + -
2 Flavonoids + + + +
3 Alkaloids - - - +
4 Tannin ++ - - ++
5 Terpenoids + + + +
6 Glycosides + + + +
Key; ++ = Highly present” + = Moderately present” - = absen
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178 Figure 2: Qualitative evaluation of phytochemicals compounds in various cultivars of crataegus
rhpidophyllum plants
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179 3.2: Comparative analysis of antimicrobial activity
Comparative analysis of antimicrobial activity of plant sample of having two fractions of methanol and hexane showed in Table 2, Figure 3. After analysis we finally concluded that both fractions of methanol and hexane was found active against selected bacterial strains of E.coli, shigilla, S. aereus, salmonella typhae and pseudomonas aeruginosa except hexane that was only inactive in hexane fraction against E.coli. Methanol and hexane fractions were found very active against S. aereus and salmonella typhae that are shown in Table 2.
Table 2:
Comparative antimicrobial activity of plant sample
S.No Bacterial Strains Zone of inhibition (mm)
Methanol Hexane
1 E.coli 13±0.89 0±0.00
2 Shigilla 6±0.56 11±0.96
3 S. aereus 12±0.71 15±0.99
4 Samonellatyphae 15±0.85 13±0.37
5 Pseudomonas aeruginosa 7±0.42 14±0.65
Figure 3: Antibacterial analysis
3.3: Mineral analysis
Analysis of mineral of plant material was determined by atomic adsorption spectrophotometer. 5 different types of mineral quantification were analyzed. After analysis it was found that all the mineral elements were found positive and exception result was found in iron quantity which was
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180 recorded 62mg/L in concentration and lower value of elements analysis was observed in cupper having 0.063mg/L concentration as shown in Table 3.
Table 3:Mineral analysis in plant sample
S. No Name of Element Concentration mg/L
1 Copper 0.063 mg/L
2 Iron 62.26 mg/L
3 Manganese 0.448 mg/L
4 Lead 0.627 mg/L
5 Zinc 0.757 mg/L
6 Cobalt 0.419 mg/L
7 Cadmium 0.211 mg/L
8 Chromium 1.332 mg/L
3.4: Antioxidant Activity by DPPH free radical scavenging assay
Four different dilutions of plant material were used to determine the antioxidant percentage inhibition of sample by DPPH method. Inhibition activity of antioxidant by plant sample was observed positive as concentrations of plant sample were increased its percentage inhibition activity was also increased that shown in Table 4.
Table 4.Antioxidant activity of plant sample
Fractions 80 PPM 60PPM 40PPM 20PPM
DPPH DPPH DPPH DPPH
Methanol 71.87±2.76 62.62±2.42 54.21±1.97 48.56±2.42
Hexane 86.65±1.98 71.98±1.91 59.94±1.84 44.21±1.97
Ascorbic Acid 89.97±1.87 77.29±1.69 66.65±1.91 59.81±2.76
CONCLUSION
After performing this research we finally conclude that Crataegus Rhipidophylla possess positive screening of all different test i.e., alkaloids, flavonoids, terpenoids, tannin, saponin, glycosides, having a pharmalogical and medicinal significance. The present study revealed that the studied plant is good source of iron 62.26 mg/L. It was also observed that this plant is potential source of antioxidant agents and due to its high antioxidant activity. It can also be concluded that bacterial activity showed good results in methanol and n- hexane. There is evidence that those plants which possess high concentration of phytochemical that can be used to different biological properties like
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181 antimicrobial, anti-inflammatory and antioxidant properties. The current investigation will help the research as basic information for future research in manipulating the buried potential of this important plant which has not been explored so far.
References
1. Adewusi, E. A., & Afolayan, A. J. (2010). A review of natural products with hepatoprotective activity. Journal of Medicinal Plants Research, 4(13), 1318-1334.
2. Alhakmani, F., Kumar, S., & Khan, S. A. (2013). Estimation of total phenolic content, in–vitro antioxidant and anti–inflammatory activity of flowers of Moringa oleifera. Asian Pacific journal of tropical biomedicine, 3(8), 623-627.
3. Anwar, F., Tayyab, M., Haq, I., & Shah, O. U. (2021). Viral overload of COVID-19 pandemics:
Overweight people a soft target to get an infection. International Journal of Clinical Virology, 5(2), 070-071.
4. Dönmez, A. A. (2007). Taxonomic notes on the genus Crataegus (Rosaceae) in Turkey. Botanical Journal of the Linnean Society, 155(2), 231-240.
5. Ju, L. Y. (2005). Crataegus oxyacantha (aubepine) in the use as herb medicine in France. Zhongguo Zhong yao za zhi= Zhongguo zhongyao zazhi= China journal of Chinese materia medica, 30(8), 634-640.
6. Kashyap, C. P., Arya, V., & Thakur, N. (2012). Ethnomedicinal and phytopharmacological potential of Crataegus oxyacantha Linn.–A review. Asian Pacific Journal of Tropical Biomedicine, 2(2), S1194-S1199.
7. Fatima, T., Mian, A. H., Khan, Z., Khan, A. M., Anwar, F., Tariq, A., & Sardar, M. (2020). Citrus sinensis a potential solution against superbugs. Applied Nanoscience, 10, 5077-5083.
8. Kumar, D., Arya, V., Bhat, Z. A., Khan, N. A., & Prasad, D. N. (2012). The genus Crataegus:
chemical and pharmacological perspectives. Revista Brasileira de Farmacognosia, 22(5), 1187- 1200.
9. Kumar, G., Karthik, L., & Rao, K. V. B. (2013). Phytochemical composition and in vitro antioxidant activity of aqueous extract of Aerva lanata (L.) Juss. ex Schult. Stem (Amaranthaceae). Asian Pacific Journal of Tropical Medicine, 6(3), 180-187.
10. Anwarl, F., Khan, M., Rehman, N., Akbar, F., Ahmadl, S., Yousaf, M., ... & Zeeshan, M. (2021).
Review and forecasting on COVID-19 outbreak; An insight to in silico candidate drug discovery. Bioscience Research, 1198-1210..
11. Subramanian, R., Subbramaniyan, P., & Raj, V. (2012). Determination of some minerals and trace elements in two tropical medicinal plants. Asian Pacific Journal of Tropical Biomedicine, 2(2), S555-S558.
12. Anwar, F., Khan, M., Salman, M., Ahmad, S., Ullah, F., Khan, J., ... & Abbas, M. (2021).
Seroprevalence of hepatitis B virus in human population of district Buner Khyber Pakhtunkhwa Pakistan. Clinical Epidemiology and Global Health, 10, 100688.
13. Yaqub, S., Farooq, U., Shafi, A., Akram, K., Murtaza, M. A., Kausar, T., & Siddique, F. (2016).
Chemistry and functionality of bioactive compounds present in persimmon. Journal of Chemistry, 2016.
http://annalsofrscb.ro
182 14. Anwar, F., Ahmad, S., Haroon, M., Haq, I. U., Khan, H. U., Khan, J., & Shah, I. A. (2019). Dengue virus epidemics: A recent report of 2017 from district Mardan, Khyber Pakhtunkhwa province, Pakistan. International Journal of Mosquito Research, 6(1), 46-49.
15. Anwar, F., Tayyab, M., Salman, M., Abdullah, Din, M., Khan, J., & Haq, I. (2020). Dengue outbreak 2018 in district Shangla KPK; clinical features and laboratory markers of dengue virus infection. Future Virology, 15(10), 693-699.
