Identification and Quantification of Phenolic Compounds by HPLC-DAD and Antioxidant activity from Cordia Gharaf Plant

Numerous indigenous plants may grow on Pakistan's soil due to its suitable climate. A few of these are now exploited as a source of expensive pharmaceuticals with therapeutic benefits. Since 4000-5000 B.C., people have used these native herbs as medicines. The Cordia gharaf plant is one of them. There are several phytochemicals found in plants, including flavonoids, alkaloids, tannins, phenolic compounds, and others, that can protect against disease. Many phytochemicals have a broad range of biological activities that help to protect against chronic illnesses, and around the world, they have been used to treat several different human ailments. According to a World Health Organization (WHO) report, more than 80% of the world's population uses traditional medicines for treatment and maintaining their health, and many valuable medications have been extracted from these medicinal plants. Therefore, scientific investigation is necessary to make enormous profits from these priceless medicinal plants. Due to the importance of the primary and secondary metabolites produced by these medicinal plants, our purpose includes the idea of examining biologically active components from these plants. Keeping in mind the foregoing, the goal of our research is to examine the phytochemicals from the selected species of Cordia gharaf that have disease-preventive properties.


Introduction
Medicinal plants have been in use for thousands of years for the treatment of different types of diseases and a large inventory of useful drugs has been isolated from them. The most significant and abundant sources of medicines are plants. Since the dawn of civilization, these herbs have been utilised all over the world to treat a variety of human illnesses (Amit Kumar et al., 2013;Sidhu et al., 2022). Ancient Egyptians have recommended chewing willow bark since the beginning of time to treat fever and headaches. Scientists eventually proved that the bark includes a salicylic acid component, which is an active ingredient utilized in the production of aspirin, many thousands of years after it was first discovered. At the moment, we are using substances that are sourced from plants (Parkash et al., 2005). Atropine has been utilized to treat some heart issues, and it's likely that a deadly nightshade plant extract has been used to relax eye muscles (Edoga et al., 2005). Phytochemicals, or chemical substances obtained from medicinal plants, have physiological effects on people. These bioactive phytochemical elements include amino acids, alkaloids, steroids, tannins, glycosides, saponins, phenolic acids, terpenoids, flavonoids, and carbohydrates, among others (Samejo et al., 20013). These components are produced by primary or secondary plant metabolism, the latter of which comprises a variety of substances used in medicine, science, agriculture, animal health, and some other sectors. (Max, R.A. et al., 2007: Vasu, K. et al., 2009.These phytochemicals, which come from several classes of substances, have been shown to have antimicrobial properties for all sorts of microorganisms in vitro and may shield animals and people against several diseases, such as cancer, diabetes, and cardiovascular problems (Muthee, J. et al., 2016). Various phytochemicals exhibit therapeutic properties that help prevent chronic illnesses (Tiwari et al., 2016;Mangi et al., 2021). For instance, flavonoids have antimicrobial, antifungal, antidiarrheal, anti-inflammatory, and antibacterial properties, while tannins and alkaloids have antimicrobial, antidiarrheal, and anthelmintic properties; polyphenols have similar properties; coumarins have antiviral properties; terpenoids have antidiarrheal properties; polypeptides have antimicrobial properties; and lectins have antiviral properties (Siddiqui, S, et al., 2009;Krishniaah D, et al., 2009;Tiwari, P. et al., 2011). There are 300 species of Cordia gharaf, most of which are found in the tropics and subtropics of the world. It has 60 different species and grows to a height of 1 to 18 metres. More than 300 species of invertebrates as well as camels and cattle are consumed by the genus Cordia (Khan, S. et al., 2004). Despite being the size of a little tree, Cordia gharaf has a deep taproot that can reach 30 metres or more below the surface of the earth. The tree is native to Iran, Bhutan, India, Bangladesh, Afghanistan, Nepal, and Pakistan. It is widely distributed in Pakistan's Khyber PakhtunKhwan (KPK) and Sindh provinces (Malairajan P. et al., 2006). Phytomedicines, often known as plant-based medications, have been utilised for a very long time. Several plant parts, including the leaves, bark, flowers, roots, seeds, and fruits, can be used to obtain this (Mojab, F. et al., 2003). Due to the significance of this knowledge for the production of biochemical components, the study of these plants' chemical composition is extremely important (Parekh, J. et al., 2007). Additionally, this plant is used as a diuretic, and carminative, and to treat liver infections and splenic or inflammatory conditions. It is also used as an astringent for conditions including vaginal discharge. (Parekh, J. et al., 2008;Khan, S. et al., 2013).As a result of these plants' abundance of essential oils and secondary metabolites with therapeutic value, our goal includes the idea of researching physiologically active components from them.

Material and Methods Plant material and Chemicals: The different
Cordia gharaf plant parts (seed, stems, and leaves) were collected from Tandojam, Hyderabad Sindh, Pakistan in August 2020. All phenolic standards used were of the highest purity and obtained from Merck (Darmstadt, Germany).

Extraction of Free Phenolic Acids and Bound
Phenolic Acids: Free phenolic and bound phenolic acids were determined by a reported method with slight variations (Memonet al., 2012). Total Phenolic Content: The total phenolic content (TPC) of Cordia gharaf extracts was determined using the reported method Folin-Ciocalteu (FC) reagent (Memonet al., 2012;Boakye et al., 2015).

Total Tannin and Total Flavonoid Contents:
Total tannin contents (TTC) and Total flavonoid contents (TFC) were determined by a reported method with slight variations (Siddiquiet al., 2017;Laghariet al., 2011;Criagg, G.M et al., 2001). Radical scavenging activity (RSA): Free radical scavenging activities from different parts of Cordia gharaf extracts were determined by using a standard procedure (Amron and Konsue, 2018). Statistical analysis: The results were evaluated using Microsoft Excel 2013 as mean±standard deviation. Minitab Software (version 16.1.1) was operated for data analysis.

Phenolic acids profiling of various parts of
Cordia Gharaf by HPLC-DAD: At a flow rate of 1 mL/minute, standards for 20 different phenolic acids were added to a chromatographic column (Table 1), (Memon et al., 2012). Characteristically, plant materials contain either free or conjugated versions of all phenolic chemicals. Simply free phenolic acids can be extracted and identified using a variety of solvents, such as ethyl acetate, methanol, aqueous acetone, etc, without the use of any acid or base. Conjugated phenolic acids can also be extracted using the hydrolysis of various plant extracts by an acid or base. Numerous thousands of phenolic compounds have been documented in the literature (Luthria, D. et al., 2006;Madhujitha, T., & Shahidi, F. 2009), employing acidic, basic, and enzymatic hydrolysis techniques or processes, the majority of which were extracted from various dry plant materials and identified as free phenolic acids. To determine the total amount of soluble-bound, free, and insoluble phenolic acids present in Cordia gharaf extracts, dried plant material was base hydrolyzed in the current investigation. In the HPLC-DAD study of Cordia gharaf extracts, the leaves contain seven bound phenolic acids and three free phenolic acids (figs. 1 and 2), while the stem (figs. 3 and 4) and seed ( fig. 5 and 6) both contain eleven bound phenolic acids where zero free phenolic acids are present in the stem and one free phenolic acid are present in seed correspondingly. In contrast, free phenolic acids were found to be more abundant in leaves (15.98 mg.g-1) than in stems (ND) and seeds (5.33 mg.g-1), which were discovered to have lower amounts of bound phenolic acids (34.43 mg.g-1) than stems (50.42 mg.g-1) and seeds (53.23 mg.g-1). As a result, the lowest amounts of protocatechuic acid (0.92 mg.g-1) and caffeic acid (1.56 mg.g-1) as found in the stems and seed, respectively. The amount of catechin acid was larger in the extracts of the leaves (11.92 mg.g-1), stems (14.86 mg.g-1), and seeds (15.37 mg.g-1), respectively. The findings are in good agreement with the literature (Robbins, R. J. 2003). Table 2 displayed the amounts of bound and free phenolic acids present in Cordia gharaf extracts from the leaves, seeds, and stems.
Total tannins, Total phenolic content, total flavonoids, and DPPH radical scavenging activity: Table 3 lists the number of total flavonoids, total phenolics, total tannins, and radical scavenging capacity in Cordia gharaf stem, leave, and seed extracts. The results demonstrated that total flavonoid and total tannin levels were found to be higher in leaves than in stems and seeds, whereas total phenolic content and antiradical activity were found to be higher in seed extracts. Total phenolic content was found to be between 31.78-58.00 mg.g-1, total flavonoids to be in the range of 21.00-33.78 mg.g-1, and total tannins to be in the range of 20.00-40.78 mg.g-1, whereas radical scavenging activity was determined to be between 55.01-120.23 µmol.100g -1 for Cordia gharaf leaf, stem, and seed extracts. Although total flavonoid and total tannin content were found to be higher in leaves compared to stems and seeds, the seed extracts also have a higher total phenolic content and significant antiradical activity. These

Discussion
Proanthocyanidins, flavonols, and flavonol glycosides are extractable from plants using methanol and aqueous acetone, respectively. Flavonols and flavonol glycosides are also extractable from plants using water. Gallic acid esters, however, are not extractable due to their extremely high molecular weight (Ramya et al. 2011;Li, Y. et al., 2006). The phenolic acids, total phenolic, total flavonoids, total tannins, and antioxidant activity of the methanol extract of Cordia gharaf stems, leaves, and seeds were determined (Contini, M. et al., 2008). Methanol was selected as the extraction solvent for phenolic compounds due to the possibility of extracting other chemicals. Because many phenolic compounds are isomerized in sunlight (trans-cis conversion), react with oxygen in basic solution (quinone production), and react with methanol at normal temperature and pH, caution should be taken when extracting them (Singh et al., 2014). Cordia gharaf is found arch source of many important phenolic compounds which possess medicinal properties and can be used as traditional medicines because this is a local plant of Sindh which is available in every region and it can be the cheapest source of many phenolic compounds. Moreover, these important compounds are extracted from the plant and can be used in the pharmaceutical and cosmetic industry as they can be less costly phenolic compounds.

Conclusion
It is concluded that the amount of bound phenolic acids in leaves was lower 34.43 mg.g-1 than in seeds 53.23 mg.g-1 and stems 50.42 mg.g-1, while the amount of free phenolic acids was higher in leaves at 15.98 mg.g-1 and lower in seeds 5.33 mg.g-1. Total phenolic content was found to range from 31.78 to 58.00 mg.g-1, total flavonoids from 21.00 to 33.78 mg.g-1, total tannins from 20.00 to 40.78 mg.g-1, and total DPPH radical activity from 55.01 to 120.23 µmol.100g-1 for the Cordia gharaf leaves, stems, and seeds, respectively. The results showed that total phenolic content and antiradical activity in seed extracts are higher than in stem and seed extracts, whereas total flavonoid content and total tannin content were found to be higher in leaves than in stem and seed extracts. In conclusion, Cordia ghraf plant can be consider as a potential source of natural antioxidant due to the presence of various phenolic compounds. The HPLC-DAD method used in this study can be useful for the identification and quantification of phenolic compounds in other plants. Further studies are needed to explore the potential application of Cordia ghraf plant as a natural antioxidant in the food and pharmaceutical industries. Acknowledgement We are highly thankful to the Institute of