INTRODUCTION:

Lawsonia inermis L is a medicinal herb from Lythraceae family¹. This plant contains the main active compounds such as flavonoids, phenols, alkaloids, glycosides, saponins, tannins, and essential oils. Flavonoids and phenols are the active compounds which most commonly found². This plant has many properties such as antidiabetic³, antimicrobial, antioxidant, antiurolitiatic, anticancer, anticonvulsant⁴, anti-irritant, anti-carcinogenic, anti-inflammatory, analgesic and antipyretic through in vitro and in vivo testing⁵. Pharmacologically, the nail henna plant has analgesic, hypoglycemia, hepatoprotector, immunostimulant, anti-inflammatory, antibacterial, antimicrobial, anti-fungal, antiviral, antiparasitic, anti-fertility, antituberculous and anticancer⁶. Flavonoids are compounds that in the phenolic compound group and can be found in nature. One of the flavonoid derivatives that act as antioxidants is flavonol⁷. Based on the amount, flavonols are the most common flavonoids found in this plant⁸. Free radicals cause damage called oxidative damage which basically can be overcome with endogenous compounds, namely antioxidants, which are naturally present in the body. However, if the number of free radicals in the body exceeds the limit of endogenous antioxidant protection capabilities, external antioxidants are needed to help the body's protection system against free radicals⁹. Antioxidants are electron-giving chemical compounds that can reduce free radicals by breaking or stopping chain reactions caused by free radicals present in the body^10.

MATERIAL AND METHODS:

Chemicals :

Glass materials (test tubes, beakers, measuring flasks), digital scales, analytical scales, maserators, vacuum rotary evaporator set (Buchi®), magnetic stirrer, 1 L separating funnel, buchner funnel, TLC chamber, vacuum liquid column chromatography (VLC), UV lamps (254nm and 365nm wavelengths), oven (Memmert) and micropipettes, aquadest.

Reagents:

Aquadest, acetone, methanol, Chloroform, FeCl₃1%, magnesium powder (Mg), 2 N HCl, 1 N NaOH, Capillary tube, TLC Silica gel 60 GF 254, ^H₂^SO₄ ^{2M, KI, HgCl}2^,^M^g^powder^,concentrated HCl, FeCl₃, toluen, NA, PDA, concentrated^HNO₃^,
HClO₃, DPPH, Vitamin c, quercetin, ^AlCl3, KCH₃COO and some reagents such as Lieberman-buchard, Mayer, Dragendorf, Wagner.

Plant Material:

Samples of Lawsomia inermis L were taken in Lamonae Village, Wiwirano District, Konawe Utara Regency. The part used is the leaves. Determination was carried out in the laboratory of the School of Life Sciences and Technology, Bandung Institute of Technology, Indonesia.

Extraction :

The dry leaf powder sample of Lawsonia inermis leaves. which has been obtained was 3kg. The sample was extracted by maceration method using 96% ethanol solvent until completely immersed for 3x24 hours. Then the maserate was carried out by the evaporation process and obtained 584.86grams of Lawsonia inermis leaves thick ethanol extract. The yield of Lawsonia inermis leaves was obtained 19.49%.

The extraction yield was calculated by the following formula¹¹.

Weight of extract (g)

% Yield of extract = -----------------------------------X 100

Weight of powdered sample (g)

Purified Extract:

Chlorophyll contained in the sample was removed by adding methanol : water with a ratio of 1 : 1, stirring and allowed to stand for 1x24 hours. The precipitate which is chlorophyll was separated from the filtrate. The 1.3 L chlorophyll-free filtrate was partitioned with ethyl acetate in a ratio of 1:1. The EtOAc phase was then added with MgSO4 as a drying agent, and filtered. The ethyl acetate phase was concentrated with a Rotary Vacuum Evaporator so that 40 grams of EtOAc extract was obtained. Furthermore, the extract was analyzed using thin layer chromatography (TLC) to determine the separation pattern of each fraction that was separated using n-hexane eluent: EtOAc (1: 1)¹².

Determination of Purified Extract:

Identification of the quality of the ingredients and the assurance of accurate purity is a necessary step quality assurance and standardization of medicinal ingredients¹³.

Standardization Parameters¹⁴:

1. Specific parameters:

a. Extract identity (a nomenclature description includes the name of the extract, the Latin name of the plant, the part of the plant used, and the Indonesian name of the plant).

b. Organoleptic extracts (extracts organoleptic determination was carried out using the senses to describe shape, color, smell, and taste. The aim was for a simple initial introduction as subjective as possible.

c. Determination of the dissolved compound content in a certain solvent: The content of the dissolved compound determination was done by dissolving the extract with a solvent (alcohol or water) to determine the amount of the solute which is identical to the amount of the compound gravimetrically. In certain cases, it can be measured dissolved compounds in other solvents such as n-hexane, dichloromethane, methanol. The goal is to provide an initial picture of the number of content compounds.

d. Content of water-soluble compounds: An amount of 1.0g of the extract was put into a clogged flask and 25.0mL of LP water-chloroform was added (2.5mL of chloroform was put in a 1000mL volumetric flask and water was added to the limit mark). Then let stand for 24 hours while shaking many times for the first 6 hours and left for 18 hours then filtered. A total of 5.0mL of the filtrate was evaporated to dryness in a shallow, flat bottom dish that has been tared. Then the residue was heated at 105^oC until the weight remains. The percent water soluble compound was calculated against the initial extract.

e. Content of the compound dissolved in ethanol: An amount of 1.0g of the extract was put into a clogged flask and 25.0mL of ethanol (96%) was added. Then let stand for 24 hours while shaking many times for the first 6 hours and left for 18 hours. Then, it is filtered quickly to avoid evaporation of ethanol. A total of 5.0mL of the filtrate was evaporated to dryness in a shallow, flat bottom dish that has been tared. The residue is heated at 105°C until the weight remains. The percent ethanol soluble compound (95%) was calculated against the initial extract.

f. Identification of Chemical Compounds: In qualitative analysis, the presence of alkaloids, saponin, tannins, terpenoid and triterpenoid and flavonoids tested¹⁵

Alkaloid Test¹⁶:

The extracts were added with 10mL of chloroform and ammonia, then crushed strongly. The chloroform layer is pipette then filtered, then 2N HCl is added, shaken, so that the two layers were separated. The acid layer at the top was piped into the two test tubes. Each test tube was added with Meyer reagent (5g KI dissolved in 90mL water and slowly added HgCl₂ while stirring and diluting to 100mL volume) and Dragendorff reagent (mixture of Bi (NO₃)₂ in nitric acid and KI solution). The presence of alkaloids indicated by the formation of a white precipitate with Meyer reagent and an orange to red brown precipitate with Dragendorff's reagent.

Flavonoid Test¹⁷:

The extracts were added with water sufficiently and heated for 5 minutes, then filtered. The filtrate was added with a little of Mg powder and 1mL of concentrated HCl, then the solution was shaken. The presence of flavonoids is characterized by the formation of yellow, orange or red colors.

Saponin Test:

The extracts were added to sufficient water and heated for 5 minutes, after which a few drops of concentrated HCl were added. The presence of saponins indicated by the formation of foam/foam which is stable for ±15 minutes.

Tanin Test :

The extracts in the test tube were added with sufficient water and heated for 5 minutes, then filtered. The filtrate was added with 1% FeCl₃. The presence of tannins indicated by the formation of a bluish green color.

Steroid and triterpenoid Test:

The extract was extracted with ether and the ether-soluble fraction separated. The ether layer was pipette and tested with Lieberman Buchard reagent (anhydrous acetic acid : concentrated H₂SO₄ = 3: 1). Red or violet colors indicate the presence of triterpenoids and green or blue colors indicate the presence of steroids.

Non-specific parameters¹⁷:

Determination of Drying Loss:

A total of 1gram of the extract was weighed in a cup that had been heated at 105ºC for 30 minutes and weighed. Roll it out by shaking it into a layer 5mm -10mm thick and dry it at the setting temperature until the weight remains, open the lid, leave the cup closed and cool in a desiccator to room temperature, then record the fixed weight obtained.

Determination of Water Content:

Determination of water content in this study using the toluene distillation method, namely the extract was weighed as much as 5g and put into a flask, then put about 200mL of toluene P that has been saturated 18-24 hours into the flask and the tool is connected and distilled until there is separation between water and toluene.

a. Determination of acid soluble ash:

The ash content of the nail henna leaf extract was determined using the gravimetric method. Weighed as much as 2g of henna nail leaf extract into a pre-weighed crucible, then gradually annealed to a temperature of 600 ± 25ºC.

b. Determination of total ash content:

Total Ash value represents the inorganic salts adhere and or present within the drug material¹⁸. The ash obtained was boiled with 25mL of dilute hydrochloric acid for 5 minutes. The collection of the insoluble portion of the acid, filtered through an ash-free filter paper, washed with hot water. The filtered ash and filter paper were put back in the same silicate crucible, slowly annealed in a crucible (with the temperature gradually increased to 600 ± 25ºC, then weighed to a fixed weight.

c. Specific Gravity:

Use a clean and dry pycnometer. The pycnometer to be used was weighed first. The pycnometer was filled with aquadest then the temperature is set at 25°C, and weighed. Aquadest in pycnometer was removed and dried to include 5% liquid extract. The liquid extract was put into a pycnometer then set at 25°C, and weighed.

d. Determination of Microbial and Fungi Contamination:

Microbial contamination:

The extract solution was made with a 1:10 dilution by dissolving 1gram of the extract in a 10mL volumetric flask. Followed by a dilution of 1 : 100 and 1: 1000. To determine the total plate count (TPC), pipette 1 mL from each dilution into a sterile (duplo) petri dish using a different and sterile pipette for each dilution. Into each petri dish 15mL of Nutrient Agar medium which has been thawed at temperature 45°C. The petri dish was shaken carefully (rotated and shaken back and forth to the right and left) until the sample was thoroughly mixed with the extract solution. Then leave it until the mixture in a petri dish freezes. Petri dishes placed in the incubator cabinet at 35°C for 24 hours. Colony growth was recorded on each plate after 24 hours and the total plate count was determined.

Mold/Yeast Contamination:

The extract solution was made with a 1:10 dilution by dissolving 1 gram of the extract in a 10mL volumetric flask. Followed by a dilution of 1: 100 and 1: 1000. The agar medium used was Potato Dextrose Agar (PDA). PDA was thawed at 45°C, then put in 15mL petri dish, let it freeze in a cup. A total of 0.5mL of each dilution of the extract solution was piped into a sterile petri dish (spreader method) using a different and sterile pipette for each dilution. The petri dish was shaken carefully until the sample was evenly distributed on the media. Then incubated at room temperature (25°C) for 7 days, then the amount of mold and yeast/g sample determined.

e. Determination of Metal Contamination:

Determination of As, Pb and Cd levels using the Atomic Absorption Spectroscopy (AAS) method. Level determination of the three heavy metals by wet digestion. 1gram of extract was weighed and 10mL of concentrated HNO₃ was added, after that it was heated with a hot plate to half of the volume. The thick and cold extract was added with 5mL of HClO₃, then heated until the white smoke disappeared and let it cool then rinsed with aquadest and filtered into a 50mL volumetric flask. Add aquadest to 50mL. Samples were measured by means of AAS. Based on the monograph book of medicinal plant extracts, the Pb metal value is not more than 10mg/kg, the Cd metal is not more than 0.3 mg/kg, while the As metal is not more than 5µg/kg.

Determination of Antioxidant Activity¹⁹:

Preparation of comparison solutions and test solutions:

To make DPPH solution, as much as 5mg of DPPH was dissolved with methanol p.a until the volume is 50ml, then 5ml was taken and diluted in a 25ml measuring flask so that a DPPH solution with a concentration of 125ppm was obtained. Stored in a measuring flask and covered with aluminum foil.

To make acidum ascorbicum stock solution, as much as 5mg of acidum ascorbicum was dissolved with methanol p.a in a 50ml measuring flask, then 5ml was taken and diluted in a 20ml measuring flask so that a stock solution of 100ppm was obtained. To make a comparison solution, a stock solution of vitamin c was taken as much as 2.5; 5.0; 7.5; 10ml diluted with methanol p.a in a 10ml measuring flask, obtained a comparison concentration of 25; 50; 75; 100ppm.

Preparation of test solutions :

Antioxidant activity test solution, as much as 10mg of purified extract dissolved with methanol p.a. up to a volume of 25ml. The solution was taken as much as 2.5; 5.0; 7.5; 10ml. Diluted to a volume of 10ml. The test solution was obtained with a concentration of 25 ; 50; 75; 100ppm.

Determination of total flavonoid²⁰:

Quercetin mains solution was made with a concentration of 200ppm, each taken as much as 1.0; 1.5; 2.0; 2.5 and 3.0ml then diluted to a volume of 10ml and obtained a concentration of 20; 30; 40; 50 and 60ppm. The blank solution consists of 1.5ml ethanol, 0.1ml 10% aluminum chloride, 1M potassium acetate and distilled water to sufficient the volume up to 10ml. The maximum wavelength was determined by taking 0.5ml of 4 ppm standard solution, plus 1.5ml etanol, 0.1ml of 10% aluminum chloride, 1M of potassium acetate and adding up to 10ml of distilled water. The absorbance was measured at a wavelength of 350 - 500nm.

The calibration curve was made by taking 0.5ml of each standard series solution, put in a 10ml volumetric flask, adding 1.5ml of ethanol, 0.1ml of 10% aluminum chloride, 0.1ml of 1 M potassium acetate and distilled water. The solution was incubated for 30 minutes, then the absorption was measured at the maximum wavelength.

Each sample solution was prepared in concentrations of 20, 30, 40, 50 and 60ppm. Then pipette 0.5ml, put into a 10ml volumetric flask, add 1.5ml of ethanol, 0.1ml of 10% aluminum chloride, 0.1ml of 1 M potassium acetate and distilled water up to 10ml volume, shaken until homogeneous. The solution was incubated for 30 minutes. Absorption was measured by a spectrophotometer at the maximum wavelength.

RESULT:

Herbal drugs must be free of mold or insects, including visible contaminants such as gravel, rock, sand, toxic and harmful foreign matter and chemical residues. Free from animal contaminants such as insects and invisible microbial contaminants, which produce toxins, as well as potential contaminants of herbal medicines²¹. Process in standardization can achieved by pharmacognostic studies²². Standardization is a process to ensure that the final product of a drug has certain constant and predetermined parameter values. There are two factors that affect the quality of the extract, namely the biological factors of the medicinal herbs/ herbs/medicinal plants and the chemical content of the medicament¹⁰. In this study, the extract was standardized with two parameters, i.e. specific parameters and non-specific parameters²³.

Specific parameters:

The characteristics of the extract were characterized by differences morphological features²⁴_.

_{Table 1. Organoleptic Observation Results}_{of the}Lawsonia inermis L. Leaves _Purified_E_xtract

Parameters	Results
Extract Name	Purified extract Lawsonia Leaves
Latin Name	Lawsonia inermis L
Plant Parts	Leaves
Color	Reddish brown
Taste	Bitter
Shape	Thick

_{Table 2. Observation Results of Compounds dissolved in water and ethanol}

Parameters	Results	Standard
Water soluble compound	11,53%	11,53%
Ethanol soluble compound	15,75%	> 6%

Non-specific parameters :

Table 3. Analysis Results of Non-specific Parameters of Lawsonia inermis L Leaves Purified Extract

Parameters	Results	Standard
Drying Shrinkage	18,36%	< 11,00 %
Water content	8,86%	≤ 10,00 %
Total ash content	3,87%	≤ 16,6 %
Acid soluble content	0,84%	≤ 7%
Specific gravity	1,0052 g/mL	-
Microbial contamination	0,61 × 10³cfu/g	≤ 10⁴cfu/g
Mold / Yeast Contamination	0,56 × 10³cfu/g	≤ 10⁴cfu/g
Pb Metal Contamination	1,51 mg/kg	≤ 10 gr/kg
Metal Contamination Cd	0,17 mg/kg	≤ 0,3 mg/kg
As Metal Contamination	Ttd	≤ 10 µg/kg

Table 4. Phytochemical Screening Test Results of Lawsonia inermis L. Purified Extracts

Type of Testing	Results
Alkaloid Test	Positive (+)
Flavonoid Test	Positive (+)
Tannin Test	Positive (+)
Saponin Test	Positive (+)
Steroid Test	Positive (+)
Terpenoid Test	Positive (+)

Antioxidant Activity Assay:

In the results of the antioxidant activity assay using the DPPH method, DPPH is a simple method using 1,1-diphenyl-2-picrylhydrazil (DPPH) as a detection compound.

Table 5. Data on The Percentage Value (%) of Antioxidants of _Lawsonia_i_nermis_L_{. Leaves}Purified Extract

Sample	Conc (ppm)	Abs Blanko	Abs Sample	% Inhibition
Purified extract	25	0,286	0,277	3,146
	50		0.086	69,930
	75		0,09	68,531
	100		0,086	_69,930
Acidum Ascorbicum	25	0,286	0,184	35,664
	50		0,175	38,811
	75		0,173	39,510
	100		0,17	40,559

The antioxidant activity assay by making DPPH solution (1,1- diphenyl-2-picrihydazil), a stable free radical compound, which if used as a reagent is sufficiently dissolved.

Figure 1: The curve of the relationship between percent (%) of antioxidant activity and sample concentration

Figure 2: The curve of the correlation between percent (%) of antioxidant activity to the concentration of ascorbic acid

The DPPH reduction method is based on the reduction of electron donors. At the time the DPPH solution is purple in color, converge electron donor materials, the DPPH will be reduced, causing the purple color of DPPH to turn yellow from the picryl group. From the research that has been done, the purified extract of henna nail leaves is able to reduce DPPH so that it turns yellow.

The effective concentration values (% antioxidants) obtained results for consecutive samples i.e. 25ppm (3.14685314), 50ppm (69.93006993), 75ppm (68.53146853), and 100ppm (69.93006993), obtained the equation linear regression Y = 1.258X + 0.820 and the IC50 value of the Lawsonia inermis L. leaves purified extract was 39.07, which means that the higher of the %inhibition of henna nail leaves purified extract, the higher of the antioxidant activity, because the IC50 value is <50ppm.

Total Flavonoid Activity Assay²⁵:

The total flavonoid content was determined spectrophotometrically in accordance with Zou et al (2004), using querstin as a standard.

Table 6. Results of the total flavonoid content test

Extract	Weight (g)	Extract Volume (L)	Abs (nm)	Mean	Total Flavonoid Content
Purified extract	0,02	0,01	0,429	0,426	3,29%
			0,428
			0,423

Figure 3: The curve of the correlation between percent (%) of flavanoid activity to the concentration of quersetin

From the measurement results of the standard quartzetine curve, it can be seen that the higher of the concentration, the higher of the absorbance value, this indicates a straight relationship between absorbance and analyte levels. This flavonoid analysis was carried out using the AlCl₃ chlorometric method. The addition of AlCl₃ which forms a complex bond with the hydroxyl group of the flavonoid compound. This change can be seen in the sample that turns yellow where the more flavonoid content of a sample, the more concentrated of yellow color was formed. The complex formation between AlCl₃ and quarsetin was stabilized by the addition of potassium acetate.

The range of total flavonoid content based on the absorbance value was between 0.2-0.8 and the absorbance values obtained were 0.429, 0.428, 0.423, respectively. The average was entered into the line equation y = 0.0047x + 0.115 with a correlation coefficient of 0.992. The sample concentration value was 66.170 mg / L. So that the total flavonoid content was 3.29% in 20 mg of purified extract.

CONCLUSION:

The purified extract of Lawsonia inermis Leaves met the quality standard of the extract.

The antioxidant activity was very strong with an IC₅₀ value of 39.07.

The total flavonoid content was 3.29% in 20 mg of purified extract Lawsonia inermis Leaves

ACKNOWLEDGEMENT:

The author thanks to the Chancellor of Mandala Waluya University for the research facilities that have been provided, especially the Unit Head and staff of the Phytochemical Pharmacognosy Laboratory for completing this research

CONFLICT OF INTEREST:

The authors declare no conflict of interest

REFERENCES:

1. Gagandeep C, Sandeep G, Priyanka P. Lawsonia inermis Linnaeus: A Phytopharmacological Review International Journal of Pharmaceutical Sciences and Drug Research. 2010; 2(2): 91-98.

2. Hasiani VV, Ahmad I, Rijai L. Isolasi jamur endofit dan produksi metabolit sekunder antioksidan dari daun pacar (Lawsonia inermis L.). Jurnal Sains dan Kesehatan. 2015;1(4):146-153.

3. Chauhan H.V. In-Vivo Antidiabetic, Lipid Lowering and Antioxidant Activities of Methanolic Extaract of Lawsonia inermis Leaves. Research J. Pharm. and Tech. 2011; 4(5):764-767

4. Patel KM, PatelPR. Review on Lawsonia inermis Linn. : An Update. Asian J. Pharm. Tech. 2017; 7(4) : 238-250.

5. Das PK, Mondal AK. Studies on traditional ‘mehendi’ used as herbal colour with special references to its antimicrobial activity and pigment profile by TLC. International Journal of Science and Nature. 2012; 3(4):799-804.

6. Chaudhary, iqbal, Nizzam Udin, Binna Shaleh, Aslan Huma, Ajmal Khan and Parveen Sahidah. Bioactive flavonoids from the leaves of Lawsonia alba (Henna). Research Institute of Chemistry, International Center For Chemical and Biological Sciences, University of Karachi, Karachi Pakistan. 2011;04(11) 454-458.

7. Koirala, N., Pandey, R.P., Parajuli, P., Jung, H.J., Sohng, J.K. Methylation and Subsequent Glycosylation of 7,8-dihydroxyflavone. Journal of Biotechnology. 2014;184, 128–37.

8. Cartea, M.E., Francisco, M., Soengas, P., Velasco, P. Phenolic Compounds in Brassica Vegetables, Molecules, 2011; 16: 251-280.

9. Birben, E., Sahiner, U.M., Sackesen, C., Erzurum, S., Kalayci, O. Oxidative Stress and Antioxiant Defense, World Allergy Organization Journal. 2012; pp. 9 – 10, 14.

10. Husni E, Suharti N, and Atma A,P,T. 2018. Karakterisasi Simplisia dan Ekstrak Daun Pacar Kuku (Lawsonia inermis Linn) serta Penentuan Kadar Fenolat Total dan Uji Aktivitas Antioksidan. Jurnal Sains Farmasi & Klinis.512-516.

11. MadhviS.K, Sharma M, Iqbal J, Younis M,SheikhR. Phytochemical analysis, Total Flavonoid, Phenolic contents and Antioxidant activity of extracts from the Leaves of Rhododendron arboretum. Research J. Pharm. and Tech. 2020; 13(4):1701-1706

12. Juliawati L.D, Ra’idah P.N, Abdurrahman S, Hermawati E, Alni A,Tan M.I, Ishikawa H and Syah Y.M. 5,6-Dihydro-α-pyrones from the leaves of Cryptocarya pulchinervia (Lauraceae). Journal of Natural Medicines. 2020; (74) :584–590.

13. Mohapatra P., Shirwaikara A., Aswatharam HN. Standardization of a Polyherbal Formulation. Pharmacognosy Magazine 2008; 4(13); 65-69.

14. Depkes RI. 2000. Parameter Standar Umum Ekstrak Tumbuhan Obat. Dirjen Pengawasan Obat dan Makanan.Volume 1 : Jakarta.

15. Shahin K, Ajay K.M, Saluja A.K and Padhi, MM. Pharmacognostical and Physicochemical Evaluation of Anthelmintic an Ayurvedic Polyherbal Formulation: Krumighattini Tablet. Asian J. Research Chem. 2015; 8(11): 697-700.

16. Muhammad S, Gul A. K, Akhtar A, Zakir U and Fazl S. Standardization of Eclipta alba (L). Asian J. Research Chem.2011; 4(12):1825-1828.

17. Harbone, J.B., 1987. Metode Fitokimia, Penuntun Cara Modern Menganalisa Tumbuhan, Terbitan II. ITB : Bandung.

18. Pathak A.V, Kawtikwar P. S, Sakarkar D. M.Pharmacognostical and Physico-Chemical Standardization of Shatavari Churna: An Official Ayurvedic Formulation. Research J. Pharm. and Tech. 2015; 8(11):1495-1501.

19. Farnsworth, N.H. Biological and Phytochemical Screening Plant, Pharm Science, 1996; 245-246, doi: https://doi.org/ 10.1002/jps.2600550302

20. Gintingg , B, Mustanir, Hira H., Lidya S.D., Era L., Rohmat M., 2017, Antixidant Activity of n- Hexana Extract of Nutmeg Plants from South Aceh Province, Jurnal Natural, Vol.17, No.1, 2017 pISSN 1411-8513, eISSN 2541-4062.

21. Narmatha. K, Arul. P, Jeyamurugan S, Srinivasan R, Vignesh. Standardisation of Rhizome Extract of Acorus calamus Linn. Asian J. Research Chem. 2018; 11(2):423-426.

22. Thomas S. Patil DA. Patil AG and Chandra N. Pharmacognostic evaluation and physicochemical analysis of Averrhoacarambola L. fruit. J Herb Med Tox 2008; 2(2): 51-54.

23. Chang, C. Yang, M. Wen, H. and Chern J. 2002.Estimation of total flavonoid contentin Propolis by two complementary colorimetric methods.J .Food Drug A.

24. Qureshi M.S, Reddy A.V, Kumar G.S and Nousheen L. Pharmacognostic, Physicochemical Standardization and PhytochemicalAnalysis of leaves of Hydrolea zeylanica Vahl. (Hydrophyllaceae). Research Journal of Pharmacognosy and Phytochemistry.2017; 9(1): 1-7

25. Zou, Y., Lu, Y., Wei, D. 2004. Antioxidant activity o flavonoid-rich extract of hypericum perforatum L in vitro. Journal of Agriculture and food Chemistry 52: 5032-50.

Received on 22.11.2020 Modified on 28.05.2021

Research J. Pharm.and Tech 2022; 15(4):1837-1842.

DOI: 10.52711/0974-360X.2022.00309

Standardization Parameters14:

1. Specific parameters:

a. Extract identity (a nomenclature description includes the name of the extract, the Latin name of the plant, the part of the plant used, and the Indonesian name of the plant).

a. Determination of acid soluble ash:

CONCLUSION:

Standardization Parameters¹⁴: