WORLD JOURNAL OF PHARMACY PHARMACEUTICAL SCIENCES Sagar et al. World JournalAND of Pharmacy and Pharmaceutical Sciences SJIF Impact Factor 8.025 Volume 13, Issue 6, 1301-1323 ISSN 2278 – 4357 Research Article SCIENTIFIC STANDARDIZATION, QUALITY CONTROL, ANTIMICROBIAL POTENTIAL, TOXICOLOGY STUDIES HAVING EFFECTIVE THERAPEUTIC USES OF (WOODFORDIA FRUTICOSA (LINN.) KURZ) FLOWERS PART Pawan Kumar Sagar1*, S. Sajwan2 and N. Z. Ahmed3 *1,2 Drug Standardization Research Institute, (Under CCRUM, Ministry of AYUSH., Govt. of India), PCIM&H Campus, IInd Floor, Kamla Nehru Nagar, Ghaziabad, U.P., India. 3 CCRUM (Ministry of AYUSH), Govt. of India, 61-65, Institutional Area, Janakpuri, New Delhi-110058, India. Article Received on 23 April 2024, ABSTRACT Standardization and product acceptability is used to describe all Revised on 13 May 2024, Accepted on 03 June 2024 measures under taken during the manufacturing process and quality DOI: 10.20959/wjpps20246-27478 control and quality assurance of drug leading to its reproducible quality. Therefore we need to develop standard validation techniques to standardize and validate of the herbal products and formulations using QC. QA Screening Studies. The drug Woodfordia fruticosa (Linn.) Kurz) is therapeutically useful in the treatment of Anti-pyretic, *Corresponding Author Pawan Kumar Sagar Anti-pills, Anti-inflammatory, Anti-ulcer, Anti-diarrheal, Anti-Sinus, Drug Standardization Anti-Diabetes, Anti-Leukorrhea, Anti-Leprosy, Anti-hyperglycemic Research Institute, (Under activity, Anti-depressant activity, Anti-cancer activity, Antioxidant CCRUM, Ministry of activity. The single drug WF was Standardization, Quality Control AYUSH., Govt. of India), Screening studied in three different batches as per the guidelines of PCIM&H Campus, IInd Floor, Kamla Nehru Nagar, Ghaziabad, U.P., India. WHO/ AYUSH Protocols basis. Present research study is aimed to evaluate the Standardization and product quality acceptability using physico-chemical parameters; HPTLC fingerprinting as per WHO guidelines of analyzed parameters. The physico-chemical average reading data’s of every III Batches of test samples showed that the drug contain Foreign matter, w/w- (0.08 %,0.07%,0.08%), LOD/ Moisture, w/w- (6.054%,6.010% 6.320%), Total ash, w/w(5.79%,5.82%,6.93%), Acid in-soluble ash, w/w-(0.776%,0.770%,0.778%), Alcohol and water soluble extractive matter, w/v- (12.80%,12.46%, 12.38%) & (24.40%, 24.46%, www.wjpps.com │ Vol 13, Issue 6, 2024. │ ISO 9001:2015 Certified Journal │ 1301 Sagar et al. World Journal of Pharmacy and Pharmaceutical Sciences 24.50%), pH(10% solution) (4.8,4.9,4.8), the HPTLC finger prints showed various spots at 254nm, 366nm Iodine vapours and visible light (M-S reagent). The tested drug samples showed significant Antibacterial potential and Toxicology studies against certain pathogens organisms and microbial load analysis. The QC. findings revealed that the test drug was free from adulterations. The evaluated validated quality standards will be very useful for referential support, validation of the standards of WF, pharmaco-vigilance and providing the quality raw medicine to deprived human being. KEYWORDS: (Woodfordia fruticosa (Linn) Kurz), Physico-chemical quality QC and QA HPTLC fingerprinting and Unani Compound drug. IDENTIFICATION The subject of standardization of herbal drugs is massively wide and deep. There are many seemingly contradictory theories on the subject of herbal medicines and its relationship with human physiology and mental function (Yadav et al., 2011).[29] Quality Control of crude material / raw material:“Quality” is the sum of all the factors which contribute directly or indirectly to the safety, effectiveness & acceptability of the product.“Standardization” describes all measures taken during manufacturing process to obtain the desirable quality. Thus the “Quality Control” leads to reproducible of a particular product in desirable manner. “The Quality criteria for herbal drugs are based on a clear scientific definition of the raw material” It is the key step. Taxonomic identification and authentification of raw materials macroscopically and microscopically have to be evaluated. Depending on type of preparation and matrix of the finished product, physico-chemical parameters such as LOD, Ash values, Extractive values, pH, and Sap value, Acid value etc; have to be observed. Adulterations have to be checked to prove identity and purity of a raw material. Microbiological contamination and foreign materials such as heavy metals, pesticide residues, aflatoxins & radioactivity are to be tested for further quality. To prove the constant composition of herbal preparations or to obtain a reproducible product adequate analytical methods have to be applied for active principles for known or unknown to Standardization or Normalization to check the criteria of uniformity (Narasimhaji V. 2018).[20] According to the WHO, the quantity, quality, safety and efficacy data on traditional medicine (TM) are not sufficient to meet the criteria needed, so some of the major policy challenges include safety, efficacy, quality, and enlightened the perception for the use of TM. Various policy measures have been applied for a clear eyed view of the use of TM, in order to www.wjpps.com │ Vol 13, Issue 6, 2024. │ ISO 9001:2015 Certified Journal │ 1302 Sagar et al. World Journal of Pharmacy and Pharmaceutical Sciences increase its safety, efficacy and acceptability (G. Bodeker and G. Burford, 2007; Sagar et al., 2023a&b).[5][6] Historically herbal medicines have played a significant role in the management of both minor and major medical illness (Bahuguna et al., 2014).[23] In order to obtain assured quality based herbal products, care through pharmaco-vigilance and utmost care has to be taken right from the beginning i.e. proper identification of plants, season and area of collection, grading, drying, extraction, purification process and rationalizing the combination in the case of poly-herbal drugs (Patel et al., 2006; Sagar et al., 2023a&b).[5][6] The Standardization and Validation of ASU herbal Drugs is not an easy challenge as various factors influence the bio efficacy and reproducible therapeutic effects. Validation of pharmacopoeial standards by experimentation and observations provides a set of characteristics to a particular herbal medicine. Therefore, Scientific Validation of Unani Formulations is an important tool used in the standardization process. (Kunle, 2012).[27] As there is increase demand of herbs and herbal products especially Unani medicinal products, run across many problems like non-availability of good quality of raw materials, proper methodology for standardization. In consequence to ensure and develop the quality, authenticity of Unani formulations, the standardization of single as well as compound drugs on modern analytical parameter is basic requirement for drugs. Before studying phytochemical and pharmacological activity of any drug physico-chemical characteristics is necessary for it’s authenticity (Naaz A et al., 2021).[12] The quality assurance and quality control of herbal crude drugs and formulated products are important in justifying their acceptability in modern system of medicine. Hence it is required to conduct the research on drugs standardization and product validation to provide effective, curable and safe drugs to the needy mass suffering from various ailments.(Sagar et al., 2024a&b; 2023 a &b; 2022b; 2021; 2020a&b; 2017).[1][2][8][9][13][14][15] Dhataki / Dhayphool / Gul-e-Dhawa plant is a very effective medicine. According to Ayurveda, Dhataki is beneficial in the treatment of many diseases. You can benefit from Dhataki in diseases like bone disease, ulcer, fever, diarrhea and piles. Many types of medicines are made from Dhataki plant. It is such an important plant that Dhataki flowers are used in almost all Ayurvedic extracts or juices. Dhataki has been used for research and other activities for many years. Let us know in which diseases Dhataki is beneficial. (dhataki plant) is of medium height. Its average height is about 3.6 meters. It is a plant rich in medicinal properties. All parts of its root, stem bark, creeper, leaves, flowers, fruits etc. are beneficial. www.wjpps.com │ Vol 13, Issue 6, 2024. │ ISO 9001:2015 Certified Journal │ 1303 Sagar et al. World Journal of Pharmacy and Pharmaceutical Sciences Various diseases are treated with Dhataki. Dhataki plants are filled with flowers every year during January to April. At this time its leaves fall. New leaves appear in its plants between February and March. Dhataki flowers are bitter in taste, cold in nature and small in size. Dhataki flowers / Gul-e-Dhaawa are helful to joining bones (Anonymous, 2021; 2011).[10] [27] Fresh Flower and Dried Flower of Woodfordia fruticosa (Linn.) Kurz clearly shown in Fig.1.a., Fig.-1b. and Fig.2-a.b.c. respectively. Fig.-1a. Fig.-1b. Fig.-2a.b.c. Fig.-1a.- Fresh Flowers of Woodfordia fruticosa (Linn.) Kurz. Fig.-1b. and Fig.-2a.b.c. Dried Flowers of Woodfordia fruticosa (Linn.) Kurz. Common language and botanical name: Its botanical (Scientific) name is Woodfordia fruticosa (Linn.) Kurz. In botanical science it is also known by the name Syn- Woodfordia floribunda Salisb. In English it is known by the names Fire-flame bush, Red bell bush etc.[10][27] www.wjpps.com │ Vol 13, Issue 6, 2024. │ ISO 9001:2015 Certified Journal │ 1304 Sagar et al. World Journal of Pharmacy and Pharmaceutical Sciences Pharmacological activities Reported effective therapeutics uses of multipurpose medicinal potent plant Dhataki / Dhai phool / Gul-e-Dhawa (Woodfordia fruticosa (Linn.) Kurz), pharmacological activities found as Anti-hyperglycemic activity; Anti-depressant activity; Anti-inflammatory activity; Anticancer activity; Wound healing activity; Hepatoprotective activity; Anti-bacterial activity; Antioxidant activity; Anti-enteroviral activity; Gastroprotective activity; Antifertility activity; Prebiotic activity; Analgesic activity; Antipsoriatic activity; Immunostimulatory activity; Anti-asthmatic activity, successfully investigated in In-vitro or In-vivo studies of research (Sagar et.al.,2024a&b;Giri et.al., 2023).[1][2][4] Regional & Different nomenclature Hindi - Dhataki pushpa / dhaya, dhai phool, English - Red bell bush, Fire-flame bush, Sanskrit- Dhataki, Dhatupushpi, Tamrapushpi, Kunjra, Subhiksha, Bahupushpi, Vahnijwala, Urdu - Gul-e- Dhawa, Bengali -Dhaiful, Oriya- Jaliko, Daathakee, Bela, Gujarati - Dhavani, Dhavdi, Telugu - Seringi, Errapurvu, Tamil- Dhathari Jargi, Vellakkai, Nepali - Dahiri, Dhayaro, Dahahari, Punjabi – Dha, Marathi - Dhayatti, Dhaavas, Malayalam - Tatiri, Tatirippu,; Origin: Plant, Part: Flower, Form: Entire part, Appearance: Normal (Anonymous,2021;2011).[10][27] MATERIALS AND METHODS Source of data collection All the data for the present study were collected from the Regional Research Institute of Unani Medicine, Chennai (NABH and NABL accredited), Central Council for Research in Unani Medicine, Ministry of AYUSH, Government of India, Tamil Nadu, India. Collection and Authentication of the Plant Material The dried plant of Sample WF1- Woodfordia fruticosa (Linn.) was procured from an authorised drug supplier in Chennai, Tamil Nadu. The local market of Chennai, India, and authenticated by Dr. Subbiah Mangeswari, Consultant Botany, Drug Testing Laboratory, Drug Standardization Research Unit, and Dr K. Venkatesan, Assistant Research Officer (Botany), Survey of Medicinal Plants Unit, Regional Research Institute of Unani Medicine, Chennai, vide reference ID. No.-7612. and another plant Samples WF2 – W F. was procured Local Vender, Shaylampur, Delhi Market and fresh plant Sample WF3 – W F. collected from Location Chamoli forest vally, Chamoli, Herbal State- Uttarakhand India region, deposited and collected by PCIM&H Syervay of Medicinal Plant Section, PCIM&H, Ghaziabad UP, State, India and authenticated by Dr. Mukash www.wjpps.com │ Vol 13, Issue 6, 2024. │ ISO 9001:2015 Certified Journal │ 1305 Sagar et al. World Journal of Pharmacy and Pharmaceutical Sciences Kumar, Research Associate (Botany), PCIM&H, Ghaziabad UP. State India Ministry of AYUSH, Government of India. and re-authenticated by Dr. Sonali Sajwan, A.R.O. (Botany), Drug Standardization Research Institute, PCIM&H Campus, IInd floor, Kamla Nehru Nagar, Ghaziabad UP. State India, under Central Council for Research in Unani Medicine, Ministry of AYUSH, Government of India. The voucher specimen has been deposited and verified at the Herbarium of the SMPU, Botany Department, DSRI, Ghaziabad UP, State India and botanically identification and cross confirmation by Mr. Jitendar, Research Assistant (Botany), Pharmacognosy Department, PCIM&H, Ministry of AYUSH, Govt. of India, Ghaziabad UP, India, vide reference ID. No.-1027. Pharmacopoeial standard parameters Pharmacopoeial research studies such as organoleptic characters, microscopically, macroscopically and physicochemically, TLC/HPLC fingerprinting, quality control and quality assurance parameters were carried out 1. Organoleptic evaluation: Organoleptic evaluation refers to evaluation of formulation by colour, odour, taste, texture etc., using the sensory organs of our body. The organoleptic characters of the drugs samples were carried out based on the method described (Siddique et al., 1995; Sagar et al., 2024a&b; 2023 a &b; 2022b).[1][2][8][9][13] 2. Powder microscopy: Take 3-5g powder drug sample was weighed, mixed with 50ml of distill water in a beaker and warmed gently in order to make complete dispersion in water. Then mixture was centrifuged and decanted supernatant. The sediment were washed several times with distilled water, centrifuged again and decanted the supernatant. Small quantity of the sediment was taken and mounted in glycerine, out of which another small quantity was taken in watch glass and a few drops of phloroglucinol and concentrated hydrochloric acid were added, mounted in glycerine to locate lignified cells and oberverved the characters under digital microscope. (Wallis, 1987; Johansen, 1940; Anonymous,2021;2011;2009;2008;1986; Sagar et al., 2024a&b; 2023 a &b; [1][2][8][9][10][13][27][32][33][36] 2022b). 3. Physico-chemical analysis: If the water content is high the drug can easily be deteriorated due to fungus, The ash content indicates the total amount of inorganic material after complete incineration and the acid insoluble ash is an indicative of silicate impurities might be due to improper washing of the drug. The alcohol and water soluble extractive indicates the amount of bioactive chemical constituents in a given amount of www.wjpps.com │ Vol 13, Issue 6, 2024. │ ISO 9001:2015 Certified Journal │ 1306 Sagar et al. World Journal of Pharmacy and Pharmaceutical Sciences particular drug when extracted with respective solvent. Some of the useful tools in standardization of ASU herbal products such as moisture content of the powdered sample at 105ºC, ash values, acid insoluble ash, solubility in water and alcohol, pH values and bulk density and estimation of sugar etc., are useful tools were studies as per standard methods (Anonymous,2021;2011;2009;2008;1986; Sagar et al., 2024a&b;2023 a &b; 2022a&b; Kumar et al., 2021).[1][2][8][9][10][13][26][27][32][33][36] 4. TLC/HPTLC finger printing analysis: The drug samples (2gm) were soaked in chloroform and alcohol separately for 18 hours and refluxed for ten minutes on water bath and filtered through What man N0.1 filter paper. The filtrates were concentrated and made up to 10 ml in volumetric flask with respective solvents (Saxena and Yadav, 1983).[39] TLC/HPTLC finger print studies of chloroform and alcohol extracts of the drug were carried out using 1307eculariz plate precoated with silica gel 60 F254 (E. Merck) with CAMAG Linomat IV sample applicator. The chromatograms of both the extracts were taken using the solvent systems toluene: ethyl acetate (8: 2 or 9 : 1) and toluene: ethyl acetate (8 : 2 or 6 : 4) and Toluene: Ethyl acetate: Methanol (7:2:1) for chloroform and alcohol extracts respectively. The plates were dried at room temperature and observed the spots at various wavelengths. The plates were scanned at 254 nm and to record the finger print spectrum after that same plates were visualized at UV-366 nm and derivatized with spraying of vanillin-sulphuric acid reagent and heated at 105° C till appeared coloured spots (Khan et al., 2022; Sagar et al., 2024a &b; 2023 a &b; 2022b; Kumar et al., 2021 and Wagner and Blad, 1996; Sethi, 1996).[13][17][26][27][32][33][36] Toxicology analysis studies 5. Estimation of microbial Load and Determination of antimicrobial activity: The microbial load viz. total bacterial count (TBC), total fungal count (TFC), Enterobacteriaceae, Escherichia coli, Salmonella spp and Staphylococcus aurous were estimated as per standard method (WHO, 1998). As well as Antimicrobial positional investigated in WF. various Flowers extraction concentrations as per applied standard methods (Sagar et al., 2024a&b; Giri et al.,2023; Najda et al.,2021; Joshi et al.,2019; Birajdar et al.,2014; Dubey et al.,2014; Sagar et al.,2006).[1][2][4][11][16][21][23][34] 6. Estimation of heavy metals: The method used for the analysis of heavy metals like lead, cadmium, mercury and arsenic as per Guidelines of WHO. Heavy metals were analyzed by Atomic Absorption Spectroscopy (Anonymous, 1998) and AOAC (Anonymous, www.wjpps.com │ Vol 13, Issue 6, 2024. │ ISO 9001:2015 Certified Journal │ 1307 Sagar et al. World Journal of Pharmacy and Pharmaceutical Sciences 2005). Details of the Instrument and operating parameters Thermo Fisher M Series, 650902 V1.27 model Atomic Absorption Spectrometer (AAS) was used for the analysis (Sagar et al., 2024a&b;2023 a &b; 2022b; Anonymous,2021;2011;2009;2008; Khan et al.,2022; Kumar et al.,2021).[13][17][26][27][32][33][36] 7. Analysis of aflatoxins: Aflatoxins B1, B2, G1 and G2 were analyzed as per Official Analytical Methods of the American Spice Trade Association (ASTA), 1997. Aflatoxins were estimated by Kobra cell techniques using Agilent HPLC and CAMAG or Anchrom HPTLC instruments as per the method ASTA. Details of instrument and operating parameters High Performance Liquid Chromatography (Thermo Fisher) and CAMAG or Anchrom HPTLC were used for the analysis of aflatoxins. Column – Ultra C18, 250 X 4.6 mm, 5 μm particles; Mobile phase: Water: Acetonitrile: Methanol (65: 22.5: 22.5); Flow rate: 1 ml/min; Temperature: 35º C; Detector: Fluorescence detector at 360 nm; Injection run: 20 μl (Aflatoxins B1, B2, G1 and G2 mixture and test samples) (Sagar et al., 2024a&b;2023 a &b; 2022a&b; Khan et al.,2022;Kumar et al., 2021; Sagar et al., 2020 a&b; 2017; 2015; 2013; Anonymous, 2011; 2009; 2008).[1][2][5][6][7][8][9][13][14][15][20][25][27][32][36] 8. Analysis of pesticide residue: The method used for the analysis of pesticide residues was as per AOAC (Anonymous, 2005). Pesticide residues were analyzed by Gas Chromatography Mass Spectra (GC-MS) (Instrument- Thermo Scientific, Model – TSQ9000 or Agilent), detector-mass selective detector or Triple Quadrupole mass analyzer detector, column specification-DB-5MS or TG-5MS, carrier gas – helium, flow rate – 1ml/min, column length – 30 m, internal diameter – 0.25 mm, column thickness 0.25 ìm).The usage of ASU. herbal products along with higher safety margins, WHO has taken necessary steps to ensure quality assurance and quality control parameters with the modern techniques and application of suitable standards, (Sagar et al., 2024a&b;2023 a &b; 2022a&b; Khan et al., 2022; Kumar et al., 2021; Sagar et al., 2020a&b; 2017; 2015; 2013; Anonymous, 2011; 2009; 2008).[1][2][5][6][7][8][9][13][14][15][20][25][27][32][36] Details Graphical Illustration / Graphical Abstract of W. fruticosa (Linn) Kurz clearly Shown as follows in Fig.-3 respectively: www.wjpps.com │ Vol 13, Issue 6, 2024. │ ISO 9001:2015 Certified Journal │ 1308 Sagar et al. World Journal of Pharmacy and Pharmaceutical Sciences Fig. 3: Graphical Illustration. RESULTS AND DISCUSSION Macroscopic: The Flowers of Dhataki / Gul-e-Dhawa (Woodfordia fruticosa) is about 1.2 cm long and occurs as single or in bunches. The calyx is about 1.0-1.6 cm long, ridged and glabrous, bright red when fresh, but on drying, it fades. Dried flower of Dhataki /Gul-eDhawa are yellowish brown in colour, innumerable, arranged in dense auxiliary paniculatecymose cluster, with short glandular pubescent pedicels. The calyx is long, striated, covered with glandular dots. The petals are yellowish brown, papery, slightly longer than the calyxteeth, ellipsoid and membranous, usually splitting the calyx near the base, and are irregularly dehiscent. A very minute sepal is attached outside the juncture of the calyx tooth and is deeper in colour. The petals are 6 in number and are stuck inside the mouth of the calyx tube, slightly longer than the calyx tooth. The filament is filiform, curved at the apex, keeping anthers inside calyx-tube, and is almost rounded or ovate. The carpels are united in 2, ovary superior, style filiform, long than ovary and stamens. (Sagar et al., 2024a&b; Giri et.al.,2023; Anonymous.2021; 2011;2009; 2008).[1][2][4][10][27][32][33] Microscopic identification: The transverse section of sepal shows a single-layered cuticularized epidermis, provided with both glandular and covering trichomes. The epithelial is multicellular, long and is consisting of a thin-walled stalk and a globose. The unicellular thick-walled is broad at the base and pointed at the apex. The ground tissue consists of thinwalled parenchymatous cells provided with sparsely distributed covering trichomes. The www.wjpps.com │ Vol 13, Issue 6, 2024. │ ISO 9001:2015 Certified Journal │ 1309 Sagar et al. World Journal of Pharmacy and Pharmaceutical Sciences transverse section of filament shows an epidermis composed of single-layered tangentially elongated cells and is covered with a very thick-cuticle ground tissue that consists of thinwalled parenchymatous cells with intercellular spaces and is surrounded by a vascular cylinder of spirally thickened vessels. The transverse section of anther shows a single-layered epidermis covered with a cuticle followed by several layers of thickened cells and is surrounded by pollen-sacs having numerous pollen grains measuring approximately 12-16 µ. The ovary is bicarpellary and laterally flattened and as such appears elongated in tranverse section. The anther lobes are tetra-sporangiate and the walls separating the locules get disorganized. A lobe shows an epidermis formed of large colourless cells followed by a fibrous layer. (Sagar et al., 2024a&b; Giri et.al.,2023;Anonymous.2021; 2011;2009; 2008).[1][2][4][10][27][32][33] Physicochemical standardization: The organoleptic evaluation of the whole plant of T E. revealed that it was Light bright golden yellow, Characteristic taste, had a Indistinct odor and Foreign Matter. (Table 1,entry 1- 4). The entire T E. flower part of plant organoleptic characteristics was discovered to be the same as those mentioned in botanical literature. Foreign substances including other plants, mould, insects, excrement, sand, stones, chemical residues, etc. are prohibited in herbal medicines. In the present study, the foreign matter in the whole plant of T E. was found to be Nil, which is within the permissible limits with reference to the Ayurvedic Pharmacopoeia of India (Ali et al.,2016; Anonymous,1986; 2000;1991; 2007). The moisture content in any herbal drug is recommended to be up to 10% (Sumbul et al., 2012), thus preventing spoilage. The Foreign matter, w/w and Loss of weight on drying (LOD/ Moisture %, w/w at 105 °C in T E. Samples WFl-1, WFl-2 and WF-3 were found to be Foreign matter, w/w- (0.08 %,0.07%,0.08%), LOD/ Moisture ,w/w(6.054%,6.010% 6.320%), The ash value is an important parameter for identifying adulterants in an herb (Ali et al., 2016). The higher ash value shows the presence of inorganic substances in the tested plant material (Husain et al., 2012). The total ash and acid-insoluble ash % values of T E. were found to be Total ash, w/w- (5.79%,5.82%,6.93%), Acid insoluble ash, w/w-(0.776%,0.770%,0.778%), respectively. The extractive values % of ethanol, and water were found to be Alcohol and Water soluble extractive matter, w/v(12.80%,12.46%, 12.38%) & (24.40%,24.46%, 24.50%) respectively. Such results indicate that most of the phytoconstituents of T E. are soluble in ethanol and water. The pH of the test material was found to be (4.8,4.9,4.8), (Table 1, entry 4–10) (Sangeeta et al., 2016). The acidic nature of the test drug shows its good absorption through the mucous membrane of the www.wjpps.com │ Vol 13, Issue 6, 2024. │ ISO 9001:2015 Certified Journal │ 1310 Sagar et al. World Journal of Pharmacy and Pharmaceutical Sciences stomach (Hardman et al., 2001). Contaminants of heavy toxic metals in plants may cause serious health issues in humans (Sangeeta et al., 2016 and Kancherla et al., 2023). The entire T E. plant’s physicochemical constants were all with in acceptable limits according to the Indian Ayurvedic Pharmacopoeia and Indian Unani Pharmacopoeia. (Sagar et al.,2024a&b; Giri et al.,2023; Sagar et al., 2022a&b; Najda et al.,2021; Sagar et al., 2020a&b Joshi et al.,2019; Dubey et al.,2014; Birajdar et al.,2014;).[1][2][4][7][8][11][13][14][16][21][23][27][32][33] TLC / HPTLC Finger Printing analysis TLC/ HPTLC finger printing profiling of alcohol extract of 2g of sample with 20ml of alcohol separately and reflux on water bath for 30min. Filter and Concentrate the filtrate up to 10 ml (approx.) on water bath and apply the alcohol extract was spotted on silica gel “G” plate / precoated aluminium TLC plate of silica gel 60 F254using HPTLC automatic sample applicator. Develop the plate in Toluene: Ethyl acetate: Methanol (7:2:1) as mobile phase, solvent system. Allow the plate to dry in air and examine under UV (254nm) shows three spots at Rf values 0.04, 0.08, 0.19 (All black); and shows seven spots under UV 366nm at Rf values 0.05 (Light blue), 0.46 (blue), 0.49 (Light green), 0.55 (blue), 0.60(blue),0.74(Light blue) and 0.83(Light blue); under Iodine vapours shows seven spots at Rf values 0.04, 0.08, 0.19, 0.37, 0.58, 0.82, 0.99 (All brown); and under visible region after derivatizing with 5% Methanolic sulphuric acid and heating the plate at 1050C for five minutes shows four spots at Rf values 0.04 (Green), 0.62 (pink), 0.71 (Grey), and 0.83(Grey). HPTLC finger printing profiling showing in Table-2 respectively. Quality Assurance and Quality control parameters Detection and validation of Pharmacopeial quality parameters of test samples in order to assess the quality of drug samples. The analysis of microbial load present in the drug showed that the total bacterial count (TBC) and total fungal count(TFC) was revealed 600 and 500 cfu/gm. The detection of the microbial load was under the permissible limits of WHO guideline. the estimation of microbial load viz. total bacterial count (TBC), total fungal count (TFC), Entherobacteriaceae, Escherichia coli, Salmonella spp and Staphylococcus aurous were analyzed and found to be in permissible limit. The results are shown in (Table - 3). The heavy metal such as lead was present within the permissible limit where as cadmium; mercury and arsenic were not detected from the drug samples. The results are shown in (Table- 5). The studies of other parameters like estimation of afltoxins such as B1, B2, G1 and G2 The results are shown in (Table- 6) and pesticide residue such as organo chlorine www.wjpps.com │ Vol 13, Issue 6, 2024. │ ISO 9001:2015 Certified Journal │ 1311 Sagar et al. World Journal of Pharmacy and Pharmaceutical Sciences group, organo phosphorus group, alachlor, aldrin, chlordane, DDT, endosulfan, heptachlor, lindane and malathion etc. were not detected from the drug, The results are shown in (Table 7) respectively. (Sagar et al.,2024a&b; Giri et al.,2023; Sagar et al., 2022a&b; Najda et al.,2021; Sagar et al., 2020a&b Joshi et al.,2019; Dubey et al.,2014; Birajdar et al.,2014;).[1][2][4][7][8][11][13][14][16][21][23][27][32][33] 9. Antimicrobial activity: The anti -microbial activity is an property of the substance to kill or prevent the growth of the bacteria it significantly increases the shelf life of the product, reduce the risk of contamination. It is an significant aspect for the quality and stability of the product. The antimicrobial activity of the formulated WF. Samples was done by from the applied appropriate Method, for the preparation of flowers extracts of W.F. the flowers were first washed 2-3 times with tap water and then with sterilized distilled water. 100 gm. Flowers of (Woodfordia fruticosa (Linn) Kurz).were crushed in blender resulting in the formation of a paste, which was mixed in 250 ml of absolute alcohol (Sagar et al.,2006; Ghanaksha and Kaushik, 1999) [34]. Alcoholic extract so prepared was allowed to evaporate at room temperature until 80 ml of it was left. This extract was squeezed through double layer musline cloth and filtered through Whattman filter paper No-42 and was centrifuged at 5000 r.p.m. for 20 minutes and then sterilized by passing through 0.2 micron disposable filter. Maller Hinton Agar (Hi media No. M173) media was used to test antimicrobial activity against E. coli, S.aureus, S. typhi and K. pneumoniae by disc diffusion method (Sagar et al.,2006; Ananthanarayana and Panikar, 1996) [34]. 5 mm diameter discs are charged with appropriate concentration of the extract and standard antibiotic oxytetracycline (one unit concentration), distilled water and absolute alcohol served as control. In the applied investigated studies 100%,50%,25% Concentration of Flowers Extracts and Anti-biotic drug Oxytetracyline 1 unit concentration as a standard taken for Anti-bacterial potential research investigation of W F. The plates are then incubated at the appropriate temperature at 370 °C for the growth of the test organisms, for 24 hours. The antimicrobial activity was evaluated by measuring the diameter of inhibition zone in mm. After incubation, the plates are examined for the zone of inhibition, which are the clear areas around the well where the growth of microorganisms has been inhibited by the test substance. The diameter of the zones are measured and is used as an indicator of potency of the antimicrobial activity. Antimicrobial activity shown to be Table- 4 respectively(Sagar et al., 2024a&b; Giri et al.,2023; Najda et al.,2021; www.wjpps.com │ Vol 13, Issue 6, 2024. │ ISO 9001:2015 Certified Journal │ 1312 Sagar et al. Joshi World Journal of Pharmacy and Pharmaceutical Sciences et al.,2019; Birajdar et al.,2014; Dubey et al.,2014; Sagar et al.,2006).[1][2][4][11][16][21][23][34] Phytochemistry & Bioactive phytochemical constituents The Stem of Dhataki contains C-glycosidenorbergenin, yield gum, and betasitosterol.k. Its leaves are rich in ursolic acid, betulinic acid, woodfruticosin, lawsone, Lupeol, betulin, betasitosterol. Woodfordia fruticosa flowers are rich in Woodfordins A- D, oenotherin A. The phytochemicals present in the plant consist of both organic and inorganic chemicals, which are secondary metabolites of the plant. These chemicals have various activities which indirectly lead to the pharmacological response of the plant. woodfordins E-I and isoschimawalin A. Therefore, W. fruticosa contains numerous chemicals in it, which are phenolic, non-phenolic, flavonoids, essential oil, etc.,(Giri et.al.,2023).[4] which contain compounds like 1,2,3,6-tetra-O-galloyl-β-d-glucose, 1,2,3,4,6-penta-O-galloyl-β-d-glucose, tellimagrandin, gemin D, heterophylliin A, woodfordins A, B, C and oenothein B. In 1992 new constituents were introduced by (Yoshida et. al.,1992).[35] The flower of this plant mostly contains flavonoids (kaempferol, quercetin) and a few non-phenolic compounds like hecogenin (Khan et.al.,2019; Raghuwanshi et.al.,2019).[17][18] The plant contains various tannins, flavonoids, alkaloids, glycosides, sterols and triterpenoids (Berhoft.2010).[29] From the flower, some known and new hydrolyzable tannin constituent is isolated and the structure of that group has been identified by (Yoshida et.al.1990),[35] and plant contained other bioactive phytochemical constituents such as γ -Terpinene, Dihydrocarvyl acetate, 1Decalone (cis-trans), cis-7-Decen-1-al, Tetradecanoic acid, Palmitic anhydride, Pentadecanoic acid, Octadecanoic acid, n-Hexadecanoic acid, and 3-Decyn-1-ol, 2,6Octadien-1-ol, 3,7-dimethyl-, acetate, €-(Geranyl acetate), as well as Caryophyllene Epoxide, Cyclopropaneoctanoic acid, Cyclopropaneoctanoic acid, 2H1-Benzopyran-2-one, 2H-1Benzopyran-2-one, and gamma-elemene (Najda et.al.,2021).[11] The leaves of the W. fruticosa were observed to have polyphenolic groups such as lawsone, glucogallin, ellagic acid, gallic acid, quercetin 3-O-(6-β-galloyl)-β-d-galactopyranoside, quercetin 3-O-α-L-arabinopyranoside, methyl 3-O-methylgallate, myricetin 3-O-α-L arabinopyranoside, etc and essential oil containing α-pinene, β-selinene, γ-curcumene, germacrene-D, β-caryophyllene, etc (Joshi et. al.,2019; Kaur et. al.,2010;Dan et. al.,1984; Saoji et.al.,1972).[16][31][37][41] while the leaves contain terpenoids such as isocarveol, geraniol, citral, thymol, eugenol, geranyl acetate, linalool, thiogeraniol, lupeol, betulin, betulinic acid, www.wjpps.com │ Vol 13, Issue 6, 2024. │ ISO 9001:2015 Certified Journal │ 1313 Sagar et al. World Journal of Pharmacy and Pharmaceutical Sciences oleanolic acid and ursolic acid (Joshi et. al.,2019; Khan et.al.,2019; Raghuwanshi et.al.,2019; Dubey et.al.,2014;Dan et. al., 1984).[16][17][18][21][37] The stem of this plant contains compounds like β-sitosterol and octacosanol (Chauhan et.al.,1976).[40] Lot of research work has been done and reported on W. fruticosa extracts, isolated phytochemicals are still unexplored, which can have a potent role in drug development. Bioassay-guided isolation may be used in the future to discover the key bioactive chemicals responsible for pharmacological effects. Although W. fruticosa has a wide range of medicinal and traditional applications, there is still a scarcity of information on the exact mechanisms underlying the pharmacological activities. Therefore, extensive research on different types of phytochemicals obtained from this plant is required to determine their exact target sites, structure-activity relationships, pharmacological activities and mechanism of action for the development of safe and effective herbal drugs for better management of different diseases (Giri et.al.,2023).[4] Table 1: Chemical identification tests. Sr. Analyzed No. Parameters 1. Colour 2. 3. Odour Taste Foreign matter, w/wTotal Ash, w/wAcid insoluble ash, w/v Alcohol Soluble Extract, w/vWater Soluble Extract, w/vLoss in wt on drying at 105OC pH (10 %) 4. 5. 6. 7. 8. 9. 10. Results Standards (WHO/API/UPI) AYUSH protocols) WF-I WF-II WF-III Light bright golden yellow Indistinct Slightly Bitter Light bright golden yellow Indistinct Slightly Bitter Light bright golden yellow Indistinct Slightly Bitter 0.08 % 0.07% 0.08% 5.79 % 5.82% 6.93% As Specified As Specified (Not more than 2.0%) (Not more than 8 %) 0.776 % 0.770% 0.778% (Not more than 1 %) 12.80% 12.46% 12.38% (Not less than 10% ) 24.40% 24.46% 24.50% (Not less than 20% ) 6.054% 6.010% 6.320% (Not more than 8% ) 4.8 4.9 4.8 As Specified I.H. Table 2: Rf values of Ethanolic extract: (By HPTLC). Solvent system Toluene: Ethyl acetate: Methanol UV Light at 254nm. 0.04 (Black) 0.08 (Green) 0.19 (Green) Rf Values UV Light at Expose under 366nm. Iodine –Vapours 0.05 (Light Blue) 0.04 (Brown) 0.46 (Blue) 0.08 (Brown) 0.49 (Light green) 0.19 (Brown) www.wjpps.com │ Vol 13, Issue 6, 2024. │ ISO 9001:2015 Certified Journal M-S reagent 0.04 (Green) 0.62 (pink) 0.71 (Grey) │ 1314 Sagar et al. World Journal of Pharmacy and Pharmaceutical Sciences (7:2:1) 0.55 (Blue) 0.60 (Blue) 0.74 (Light Blue) 0.83 (Light Blue) 0.37 (Brown) 0.58 (Brown) 0.82 (Brown) 0.99 (Brown) 0.83(Grey) Table 3: Analysis of microbial load. S. N0. 1 2 3 4 5 Parameter analyzed Total Bacterial Count Total Fungal Count Escherichia coli Salmonella typhai Spp. Staphylococcus aurous Results 600 cfu/gm 500 cfu/gm Absent Absent Absent WHO Limit 105cfu/gm 103cfu/gm Absent Absent Absent Table 4: Evolution of Anti-Microbial Activity. The Antimicrobial Effect of Woodfordia fruticosa (Linn) Kurz Flower Extract Inhibition zone in mm Antibiotic Zone, (Oxytetra Cycline- 1 unit Cont,) Organism 33 32 33 20 28 Escherichia coli Pseudomonas sp. Hygrobacterium sp. Salmonella typhi K. pneumoniae Undiluted Extract Zone (A) 100% 24 Nil Nil 20 20 50% 18 Nil Nil 16 18 25% 16 Nil Nil 14 12 Control Alcohol Zone (B) Distt. Water Zone (C) mm 5 5 5 5 5 mm Nil Nil Nil Nil Nil Effective Zone of Inhibition (in mm) (A-B) 100% 19 Nil Nil 15 15 50% 13 Nil Nil 11 13 Table 5: Estimation of heavy metals. S. N0. 1 2 3 4 Parameter analyzed Lead Cadmium Mercury Arsenic Results 2.52ppm 0.03ppb Not detected 0.09 ppm WHO Limit 10ppm 0.3ppm 1.0ppm 3.0ppm Table 6: Estimation of aflatoxins. S. N0. 1 2 3 4 Parameter analyzed Aflatoxins, B1 Aflatoxins, B2 Aflatoxine, G1 Aflatoxine, G2 www.wjpps.com │ Vol 13, Issue 6, 2024. Results Not detected Not detected Not detected Not detected WHO Limit 0.5ppm 0.1ppm 0.5ppm 0.1ppm │ ISO 9001:2015 Certified Journal │ 1315 25% 11 Nil Nil 9 7 Sagar et al. World Journal of Pharmacy and Pharmaceutical Sciences Table 7: Estimation of pesticide residues. S. N0. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Parameter Analyzed DDT (all isomers, sum of ρ, ρ’DDT, α, ρ’ DDT, ρ, ρ’-DDE and ρ, ρ’-TDE (DDD expressed as DDT) HCH (sum of all isomers) Endosulphan (all isomers) Azinphos-methyl Alachlor Aldrin (Aldrin and dieldrin combined expressed as dieldrin) Chlordane (cis& tans) Chlorfenvinphos Heptachlor (sum of heptachlor and heptachlor epoxide expressed as heptachlor) Endrin Ethion Chlorpyrifos Chlorpyrifos-methyl Parathion methyl Malathion Parathion Diazinon Dichlorvos Methidathion Phosalone Fenvalerate Cypermethrin (including other mixtures of constituent isomers sum of isomers) Fenitrothion Deltamethrin Permethrin (sum of isomers) Pirimiphos methyl Results WHO Limit (mg/kg) Not detected 1.0 Not detected Not detected Not detected Not detected 0.3 3.0 1.0 0.02 Not detected 0.05 Not detected Not detected 0.05 0.5 Not detected 0.05 Not detected Not detected Not detected Not detected Not detected Not detected Not detected Not detected Not detected Not detected Not detected Not detected 0.05 2.0 0.2 0.1 0.2 1.0 0.5 0.5 1.0 0.2 0.1 1.5 Not detected 1.0 Not detected Not detected Not detected Not detected 0.5 0.5 1.0 4,0 CONCLUSION Drug Standardization, quality control, quality assurance, pharmaco-vigilance are an essential part for the evaluation and validation of scientific standards to justify the quality of herbal single drug. To maintain the batch-to-batch uniformity, consistency and quality of the drug, each plant drug material used in quality acceptance of Woodfordia fruticosa (Linn.) Kurz) WF1, WF2 and WF3 samples were confirmed, identified and evaluated for their pharmacopoeial standards. TLC/HPTLC finger print profile of alcohol extract provided a suitable method for monitoring the identity and purity and also standardization of the drug samples. In the present investigated research studies of various analyzed data, quality www.wjpps.com │ Vol 13, Issue 6, 2024. │ ISO 9001:2015 Certified Journal │ 1316 Sagar et al. World Journal of Pharmacy and Pharmaceutical Sciences standard parameters such as heavy metals, Aflatoxins, pesticide residues and microbial load were found within permissible limit of WHO guidelines. Physico-chemical, TLC/HPTLC finger printing, WHO parameters were revealed and carried out can be laid down as reference standards of the drug W F.- WF1, WF2 and WF3 samples From the present studies it can be concluded that the single W F samples is safe and free from any toxic, hazardous substance. The Antibacterial potential research data shows that various alcoholic flowers extracts concentration of W.F substantially inhibited the growth of E.coli, S. tyaphi and K. pneumoniae but not inhibited the growth of Pseudomonas sp. and Hygrobacterium sp. as indicated by size of zone of inhibition. Thus the Flower extracts of the plant samples of W.F shows broad spectrum antibacterial activity. The flower extracts of various samples was more effective against Gram +v. It is an economic drug and the efficacy of the drug can be used as a traditional alternative medicine as a treatment of Anti-pyretic, Anti-pills, Antiinflammatory, Anti-ulcer, Anti-diarrheal, Anti-Sinus, Anti-Diabetes, Anti-Leukorrhea, AntiLeprosy, Anti-hyperglycemic activity, Anti-depressant activity, Anti-cancer activity, Antioxidant activity, control and protect Gynaecology, Over bleeding, Spleen, teething related etc. problems. as mentioned in the classical Ayuvedic and Unani, authenticated and AFI Pharmacopeial literature or text basis. Can be incorporated of pharmacopoeial standard monograph. further studies are expected to advance comprehend confirmation the In-vivo detailed mode of action upon animal model of its dynamic active polyherbal active phytochemical constituents based classical multipurpose herbal raw traditional medicine potent therapeutics and to completely reveal its preventive and healing potentials. Ethical approval As the work is purely an in-vitro study, ethical clearance is not required. Author contributions Dr Pawan Kumar Sagar (Chemistry): Carried out Instrumental, Chemistry part and Manuscript written. Dr N Zaheer Ahmed (Unani): Unani expert, Work designed and revised manuscript review, supervised. S. Kashyap (Chemistry): Analytical data analysis. Declaration of Conflict, Competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. www.wjpps.com │ Vol 13, Issue 6, 2024. │ ISO 9001:2015 Certified Journal │ 1317 Sagar et al. World Journal of Pharmacy and Pharmaceutical Sciences ACKNOWLEDGMENT The authors are extremely thankful to the Dr. N. Z. Ahmed, Director General, CCRUM, New Delhi, under ministry of AYUSH, Govt. of India for his valuable guidance, encouragement and necessary research facilities to carry out the research studies as well as also thankful to our all of dedicated research staff team of the research Institute for providing full cooperation and valuable support to complete this research work. REFERENCES 1. Sagar PK, Khan AS, Sajwan S, Kashyap S, Ahmad R. An concise overview on standardization research of ASU-TAM herbal formulated and single drugs, products, 2024a; 6(1): 86-95. DOI: 10.33545/26647222.2024.v6.i1b.91 2. Sagar PK, Khan AS, Sajwan S, Ahmad R. शोध रेख - धातकी / धाई पूर / गुर-ए-धावा के प्रबावी चिककत्सीम उऩमोग वारे वैज्ञाननक भानकीकयण, गुणवत्ता ननमंत्रण स्क्रीननंग अध्ममन( वड ु पोर्डिमा फ्रुटिकोसा( लरनन ).कुर्जि) International Journal of Applied Research, 2024b; 10(5): 39-53. DOI:10.22271/allresearch.2024.v10.i5a.11729 3. 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