The leaves of Cassia alata were collected from four different locations in Indonesia: Bogor Botanical Garden, West Java (6°35'51.0"S, 106°47'58.4"E), Bogor, West Java (6°23'03.3"S, 106°40'46.5"E), South Tangerang, Banten (6°21'13.1"S, 106°39'31.6"E) and Banjarmasin, South Kalimantan (3°19'38.7"S, 114°35'26.8"E). The plants were identified at Herbarium Bogoriense, Department of Botany, Indonesian Institute of Sciences, Research Center of Biology.

C. alata leaves were dried and ground using a blender to get a simplicia powder. The powder was macerated with 70% ethanol, with the ratio of 1:5 (ratio of simplicia powder and solvent) and stirred by a shaker at 110 rpm for 18 h. The extraction was repeated until the macerate color resembled the color of the solvent. Then, the extract was evaporated with a vacuum evaporator at 50 °C, yielding of the thick extract (Angelina et al. 2020).

The chemical used in the study were 70% ethanol, ether, methanol, HCl, CHCl₃, H₂SO₄, HNO_3, FeCl₃, NaNO₂, AlCl_3, NaOH, H₂O_2, Na₂CO_3, NaCl, Mayer reagent, Dragendorff reagent, Bouchardat reagent, Folin-Ciocalteau, Quercetin, Gallic acid, purchased from Merck.

Microbial culture media used were Peptone Water, Potato Dextrose Agar (PDA), Selenite Cystine Broth (SCB), Luria Broth (LB), Mueller-Hinton Agar (MHA), and Brain Heart Infusion (BHI), purchased from Himedia.

The organoleptic test was carried out to observe the shape and color of simplicia and extract according to (Depkes RI 2000).

Physicochemical parameters include loss on drying, water content, total ash content, acid-insoluble ash content, water-soluble ash content, water-soluble extract content, and ethanol-soluble content.

LOD was determined by gravimetric determination. 2–5 g of sample was placed in crucible porcelain, dried at 105 °C for 60 min, then moved into a desiccator. This process was repeated until the constant weight was achieved. LOD was expressed as gram per gram of air-dried (World Health Organization 1998).

Water content was determined by gravimetric method. 1 g of sample was heated in the oven at 105⁰C for 5 h, and then weighed. The process was continued with 1 h intervals until the difference between 2 consecutive weighings is not more than 0.25% (Depkes RI 2000).

1 g of sample was placed in a silicate crucible and weighed. Sample was spread in an even layer in the crucible, and the material ignited by gradually increasing the heat to 500–600 °C until free from carbon, cooled in a desiccator, and weighed. Repeatedly until a fixed weight is obtained (World Health Organization 1998).

Ash obtained from ash content testing was boiled with 25 mL HCl (~70 g/l) TS for 5 min. The ash is filtered with non-ash filter paper and washed with 5 mL hot water. The insoluble matter was transferred to the crucible, dried on a hot-plate and ignited to constant weight, and placed in a desiccator for 30 min, then weighed without delay. Content of acid-insoluble ash is calculated in mg per g of air-dried material (World Health Organization 1998).

Containers containing total ash were added with 25 mL of water and boiled for 5 min. Material that does not soluble is collected into a glass cup or ashless filter paper. Then, it was washed with hot water and ignited in a cup for 15 min at a temperature of 450 °C until the weight remained. The reduction of the residue weight in mg is total ash weight. The water-soluble ash is calculated in mg per g of air-dried material (World Health Organization 1998).

5 g of extract was macerated with 100 mL of water for 6 h for water-soluble extract determination, and then saturated with CHCl₃. For ethanol-soluble determination, it was macerated with ethanol. They were shaken frequently, and allowed to stand for 18 h. The extract produced was filtered and poured into a volumetric flask. 20 mL of extract was transferred to a porcelain cup, evaporated until dry. The residue was heated using an oven at 105 °C to receive constant weight. The soluble extract was calculated in g per g of air-dried material (Depkes RI 2000).

The results of organoleptic observations from shape and color of simplicia and C. alata leaf can be seen in Fig. 2 and Table 1. The data obtained are used as a standard for good simplicia raw materials and extracts before being used for further testing. The simplicia of C. alata leaf from Bogor, Bogor Botanical Garden, South Tangerang and Kalimantan had a rough shape with a brownish green color. While the 70% ethanol extract was thick and brownish black in color.

Table 2 shows the result of physicochemical analysis of C. alata simplicia and extracts. The loss of drying (LOD) determines the maximum limit range of the compound lost during the drying process. During the drying process, the air and compounds with a lower boiling point than air will evaporate (Depkes RI 2000). According to the results, losses on drying from simplicia from the lowest were C. alata from Kalimantan 3.93%, Bogor 5.09%, Bogor Botanical Garden 5.15%, and South Tangerang 5.47%. While losses on drying of extract from the lowest were Kalimantan 8.51%, Bogor 9.35%, South Tangerang 17.38%, and Bogor Botanical Garden 19.76%, respectively. The LOD value of the extract we examined was higher than the previous study (Gritsanapan and Mangmeesri 2009) whose LOD value was no more than 6%. One of the causes is due to the difference in solvents in the extraction process. In this study we used ethanol 70% while in the previous study used ethanol 80%. The high water content in the solvents we use is one of the things that cause the extract’s LOD value to be higher.

Determination of water content in the sample was set to maintain the quality of the extract. The less water content in crude and extracts, the less possibility ofcontamination by mold growth. According to the literature (Depkes RI 2008), the water content should not be more than 10%. The water content of simplicia is between 2.12%–4%. Hence, it can be concluded that the water content of all samples of simplicia C. alata leaf met the standard.

Determination of total ash content was carried out to measure the total amount of external and internal mineral in simplicia or extract from the initial process to the end of manufacture. The high value of the total ash content indicates that the sample contains minerals. Total ash content in simplicia was 5.4%–9.41% and in extract was 6.94%–17.07%. In this test there are two parameters to determine type of nutrient absorbed by plants, acid-insoluble ash content and water-soluble ash content. Water-soluble ash content was tested by dissolving the ash into water, and determined the amount of mineral soluble in water, while the acid-insoluble ash content indicates the amount of mineral insoluble in acid. The high value of acid-insoluble ash content indicates that the sample has a silicate component associated with soil or sand, silver, lead or mercury (Salim 2016). Acid-insoluble ash content in simplicia was 0.29%–5.41% and in extract was 0.52%–3.82%. Water-soluble ash content in simplicia was 1.26%–7.14% and in extract was 1.43%–8.54%. Based on the results, the value of water-soluble ash content is higher in simplicia and extract indicates that the sample does not contain much silica component or other impurities.

The soluble content parameter determines the amount of chemical components in simplicia and extracts in certain solvents. The chemical compounds in the sample which can be dissolved are related to reproducibility in the pharmacodynamic activity of the crude drugs. The ability of each plant to dissolve in water or other organic solvents is different. Water solvent used to dissolve polar compounds and ethanol to dissolve the less polar compounds (Wirnawati et al. 2020). Water-soluble content in simplicia was between 19.60%–39.43%, and in extract was 58.45%–77.51%. The ethanol-soluble content in simplicia was 13.99%–33.76% and in extract was 59.79%–75.39%. Based on the results (Table 2) water-soluble content value in simplicia and extract is higher than the levels of ethanol-soluble content, which indicates that the sample of simplicia and extract contain more polar substances which make it easier to dissolve in water than ethanol.

Figure 1. 

Cassia alata leaves collected from South Tangerang, Indonesia.

Figure 2. 

(A–D) The organoleptic profiles of C. alata simplicia. A: Bogor; B: Bogor Botanical Garden; C: South Tangerang; D: Kalimantan. It had rough shape with a brownish green color. (E, F) The organoleptic profiles of C. alata ethanol extract. E: Bogor; F: Bogor Botanical Garden; G: South Tangerang; H: Kalimantan. It was thick and brownish black in color.

Phytochemical screening was carried out to identify the phytoconstituents present in the C. alata leaf extract. Based on the screening (Table 3), the compounds contained in the ethanol extract of C. alata leaves from Bogor, Bogor Botanical Garden, South Tangerang, and Kalimantan are alkaloid, flavonoid, terpenoid, saponin and tannin. The results obtained are in accordance with the research conducted by (Kumavat et al. 2011) which showed that the phytochemical components contained in the ethanol extract of C. alata leaves were alkaloids, tannins, steroids, glycosides, sterols, terpenoids, flavonoids. As well as research conducted by (Raji et al. 2015; Nagarajan 2018) which showed that the content of phytochemical compounds in the ethanol extract of C. alata leaves were phenols, steroids, flavonoids, terpenoids and alkaloids.

Alkaloids are semi polar, have a nitrogen atom that has a lone pair of electrons that will bond covalently to the iodine ion in the reagent and have substituents such as phenol, amine, amide and methoxy (Kurmukov 2013). Alkaloids qualitative tests were carried out using Dragendorff, Meyer and Bouchardat reagents. The results showed the presence of alkaloids with the formation of orange sediment in Dragendorf’s reagent and brown sediment in Bouchardat’s reagents, while in Meyer’s reagents there was no sediment. Terpenoids consist of several compounds including volatile monoterpenes, sesquiterpenes, volatile diterpenes, non-volatile triterpenes and sterols. It is characterized by the formation of a red, pink or violet color. The results showed that the extract of C. alata leaf contained terpenoids. Saponin is a secondary metabolite belonging to surface compounds and found in many plants. Saponins are characterized by the presence of foam after shaking in aqueous solution. The results showed that the extract of C. alata leaf contains saponins. Tannins are compounds that are commonly found in vascular plants, have a phenol group and astringent taste (Kurmukov 2013). Based on the results, the extract of C. alata leaf contained tannin compounds. This was indicated by a change in the color of the solution to blackish green.

The measurement data using LC-MS is seen at Table 4 and the chromatograms are shown in Fig. 3. The chemical constituents detected were emodin, kaempferol, kaempferol-3,7-diglucoside, and kaempferol-3-O-β-D-glucopyranoside (Fig. 3). It can be seen that the % area detected from each compound from four sources in Indonesia was different, hence it can be concluded that the phytochemicals of extracts from different locations were different.

From our previous study (Angelina et al. 2020), the ethanolic extract of C. alata leaf contained 5,7,2’,5’-tetrahidrox-flavone, daturametelin H, and kaempferol-3,7-diglucoside. Metabolic profiles of medicinal plants are mainly controlled by genetic factors, but their production is also influenced by environmental factors (Muraina et al. 2008; Mohammadi Bazargani et al. 2021). In another studies, it has been reported that chemical constituents of C. alata leaf ethanol extract were aloe emodin, emodin, ω-hydroxyemodin, lunatin, physcion, ziganein, apigenin, 7,4´-dihydroxy-5-methoxyflavone, diosmetin, kaempferol, luteolin, trans-dihydrokaempferol, trans-resveratrol (Promgool et al. 2014). From numerous studies, flavonol compounds which have been identified were kaempferol, kaempferol-3-O-β-D-glucopyranoside, 3,5,7,4’-tetrahydroxy flavone, 2,5,7,4’-tetrahydroxy isoflavones, anthraquinone and kaempferol 3-O-gentiobioside (Fatmawati et al. 2020). Alkaloid compounds from C. alata leaves have also been identified, such as adenine, chrysoeriol, quercetin, 5,7,40-trihydroflavanone, kaempferol-3-O-beta-D-glucopyranosyl-(1→6)-beta-D-glucopyranoside, n-dotriacontanol, n-triacontanol, stearic acid, palmitic acid, diomestin, luteolin (Joel et al. 2017), and 1,3,5-trihydroxy-7-methylanthracene-9,10-dione (Promgool et al. 2014; Joel et al. 2017; Fatmawati et al. 2020).

The total flavonoids content (Table 3) ranged from 9.86% to 15.74% QE. The highest concentration was found in C. alata from Kalimantan (15.74%) followed by Bogor (14.13%), Bogor Botanical Garden (10.5%) and South Tangerang (9.86%). Quantitative analysis of total flavonoid compounds was carried out with measurements based on the formation of complexes between AlCl₃ with ketone groups on C-4 atoms and hydroxyl groups on C-3 or C-5 atoms adjacent to flavones and flavonols (Nurlinda et al. 2021). The standard solution in this study was quercetin, a flavonol that is widespread in plants, and made up to 60–75% of flavonoids (Nurlinda et al. 2021).

The concentration of total phenolic content is given in terms of GAE/g of the extract (Table 3) and ranges from 6.67 to 7.65% GAE. The highest amount of phenolic content was observed in C. alata extract from South Tangerang (7.65%), followed by Bogor (6.75%), and botanical garden (6.67%). Phenolic compounds are widely distributed in a variety of plants. Natural phenolic can range from simple molecules, such as phenolic acid, to highly polymerised compounds, such as tannins (Debebe et al. 2016). The total phenolic content of the extract C. alata leaf was determined using Folin-Ciocalteu’s reagent. Phenols reacted with the Folin-Ciocalteu reagent in the presence of sodium carbonate to form a blue-coloured complex. The intensity of the blue color is proportional to the amount of reactive phenolic compounds in the sample (Kupina et al. 2019). It is also mentioned in (Debebe et al. 2016), that the presence of compounds in plants is largely influenced by genetic factors and environmental conditions, other factors such as germination, degree of ripeness, variety, processing and storage. Based on the results obtained, it shows that the area where C. alata grows affects the total amount of flavonoids and phenolics.

Figure 3. 

(A–D) The chemical constituents detected in C. alata leaf ethanol extract. A: Emodin. B: Kaempherol. C: Kaempferol-3-O-β-D-glucopyranoside. D: Kaempferol-3,7-diglucoside). (E–H) The chromatograms from LC-MS of C. alata leaf ethanol extract. E: Bogor. F: Bogor Botanical Garden. G: South Tangerang. H: Kalimantan. The chemical constituents are marked by green arrow: kaempferol-3,7-diglucoside; red arrow: kaempferol-3-O-β-D-glucopyranoside; yellow arrow: kaempferol, blue arrow: emodin.

Heavy metalcontamination testing aims to determine the levels of metal content, i.e. Pb, Cd, Hg and As contained in extract, which is dangerous and toxic to the body. The results obtained on the levels of Pb, Cd, Hg and As shows the absence of heavy metals in C. alata leaf extract (Table 5), hence the samples meet the standards, where the maximum limit for Pb is ≤ 10 mg/kg, Cd ≤ 0.3 mg/kg, Hg ≤ 0.5 mg/kg and As ≤ 5 mg/kg in herbal capsule containing dry extract (Vinet and Zhedanov 2011).

Microbial contamination reveals the impurity in medicinal plants, which come from the preparation or final products. Based on microbialcontamination test, the total plate number shows the absence of bacteria in the extract of C. alata leaf (Table 5), hence the samples meet the standard of (BPOM 2014) which shows that the maximum limit range for the total plate number test is ≤ 10⁵ CFU/g.

The results of total yeast and mold number test extract of C. alata leaf from Bogor, South Tangerang and Kalimantan did not contain yeast and mold, while extract of C. alata leaf from Bogor Botanical Garden contained yeast and mold of 1×10² CFU/g. The result was still below the limit, where the limit of mold and yeastcontamination that may be present in capsule preparations containing dry extract is ≤ 10³ CFU/g (BPOM 2014).

The identification test of pathogenic bacteria is carried out to detect the presence of pathogenic bacteria including Escherichia coli, Pseudomonas aeruginosa, Salmonella sp, and Shigella sp. Based on the test results, in all samples there were no pathogenic bacteria tested. This met the standard of (BPOM 2014) which shows that the maximum limit of E. colicontamination is ≤ 10 CFU/g, for Salmonella and Shigella negative/g.

Antibacterial activity of C. alata leaf can be seen in Table 6. From the MIC value, the extract from all areas showed similar antibacterial strength against all bacteria tested whether Gram positive bacteria (S. aureus, S. epidermidis, and B. subtilis) or Gram-negative bacteria (E. coli). The MIC of all samples against S. aureus was 250 µg/mL, meanwhile for the rest of bacteria tested the MIC was 125 µg/mL. From the MBC value, the lowest concentration is from extract from South Tangerang and Kalimantan which was 1000 µg/mL against S. aureus and 250 µg/mL against other bacteria, whilst the highest MBC was from Bogor which was 1000 µg/mL against S. aureus and 500 µg/mL against other bacteria. It can be seen that the geographical factor from the plants affects its antibacterial activity.

The antibacterial activity from this study confirms previous study (Khan et al. 2001; Somchit et al. 2003) where it reported C. alata leaves extract antibacterial activity. The MIC and MBC values against E. coli bacteria obtained in this study were similar to the values of MIC and MBC in previous studies (El Mahmood AM 2008). The antibacterial effect of the C. alata leaves extracts may be due to presence of flavonoids, phenols, alkaloids, saponins, tannins, and terpenoids detected (Wikaningtyas and Sukandar 2016). Several researches have investigated the isolated compound of C. alata which are responsible for antibacterial activity. Bioactive compounds such as kaempferol, luteolin, and aloe emodin isolated from the methanolic leaves extract showed antibacterial activity against MDR bacteria strains, which could be linked to the anthraquinone and flavonoids compounds detected (Joel et al. 2017). Compounds detected in this study also were reported have antibacterial activity. Emodin isolated from C. alata showed strong antibacterial activity against MRSA with MIC value of 4 μg/mL and moderate antibacterial activity against B. cereus and S. aureus MIC value of 16 μg/mL, but showed weak antibacterial activity against Gram-negative bacteria (MICs 128 μg/mL) (Promgool et al. 2014). Kaempferol 3-O-β-glucopyranoside exhibited MIC₅₀ values of 13.0 ± 1.5 μg/mL against MRSA (Hazni et al. 2008). Kaempferol and kaempferol-3,7-diglucoside also have been investigated to have antibacterial activity (Saito et al. 2012; Mahmood 2014). Other studies have also reported that C. alata has activity against microorganisms that cause skin diseases in human (Ibrahim and Osman 1995).