Scopoletin, galic acid, quercetin, Folin Ciocalteu’s, gallic acid, HEPES and Rabbit lung by acetone dehydrated were purchased from Sigma-Aldrich (St. Louis, Missouri, United States). Ethanol 96%, methanol, ethyl acetate, n-hexane from JT Baker. Substrate hippuryl-glycyl-glycine (Hip-Gly-Gly) was from Bachem (Torrance, CA, USA), and captopril (Cat: 1091200 US Pharmacopeial).

The ripen fruits of Morinda citrifolia (Noni) were collected from three growing different locations in Indonesia such as Tangerang, Bogor, and Sukabumi, in September 2020. Plants were identified by Botanist in the Herbarium Bogoriense, Research Center for Biologi-BRIN. Samples were washed with running tap water, drained, and chopped into small pieces. Samples were dried in an oven at temperature of 45 °C for three days and ground into powder for further analysis. Each the dried powdered of fruit (500 g) were extracted using 70% ethanol under maceration condition. The organic solvents were evaporated using rotary vacuum evaporator to produce crude extract.

The assessment including macroscopic/microscopic studies, water content, ash content, acid insoluble ash content, water soluble extract content, ethanol-soluble content, pathogen-microbial contaminant, and heavy metal contaminant were referred to the Indonesian Herbal Pharmacopeia (PHI)1^st (Depkes 2008).

Phytochemical analysis to detect the presence of alkaloid, flavonoids, triterpenes/steroid, and saponin in the extract using standard methods was carried out using colorimetric methods according to previous study with slight modification (Yadav et al. 2011). The total phenolic content was estimated according to Folin-Ciocalteu method and total flavonoid was determined according to aluminums trichloride method using quercetin as the reference compound (Dewi et al. 2019).

Each experiment was performed in triplicate. To calculate standard error and mean, Microsoft Office Excel 2019 (Microsoft Corp, USA) was used.

It has been known that pharmacognostic evaluation of medicinal plant is an important step in quality control of herbal medicine. Macroscopic and microscopic characterization as well as phytochemical analysis have been performed to identify and detect some adulteration and substitution that may be reduce purity and quality of raw materials. In this study, Noni crude drugs and its extract have been standardized based on its pharmacognostic and phytochemical analysis.

As shown in Fig. 1A, the macroscopic characterization of Noni fruit was ovoid, ellipsoid or roundish (3–10 × 3–6 cm) with an embossed appearance, slightly wrinkly, waxy, semi-translucent skin, and turns from green to yellow and to almost white as it ripens; the fruit surface is faintly patterned with 4- to 6-sided outlines, each with a central “eye”; the pulp is fleshy and juicy, dull-yellow or yellowish white and gelatinous when the fruit is ripe; it has numerous hard oblong-triangular reddish-brown pits, each containing 4 seeds about 3.5 mm long. In the form of a cross-section of the fruit, the shape of a flat slice, the outer surface is smooth with remnants of the seed burrows, the inner surface is rough, there are 4–5 fruit compartments, each partition with 2–3 seeds, the protrusions of the seeds are clearly visible, brown in color, characteristic odor, slightly bitter taste. Furthermore, for its microscopic characteristic, the crude drugs contain thin-walled parenchyma cells, exocarp and sparsely distributed thin strands of vascular tissue containing tracheary elements with annular, spiral and rarely, pitted secondary walls; acicular calcium oxalate crystals, starch granules and oil globules (Fig. 1B). The macroscopic and microscopic observations revealed that noni fruit crude drug was in agreement with the Indonesian Herbal Pharmacopeia.

Figure 1. 

Macroscopic characterization of Morinda citrifolia crude drug (A); Microscopic characterization of Morinda citrifolia crude drug powder (B); mesocarp (magnification 400X) (1); thin-walled parenchyma cells (magnification 400X) (2); endocarp (magnification 100X) (3).

Phytochemical screening provides a general overview of active compounds presence in the medicinal plants. Phytochemical compounds in a plant extract can be affected by several factors, including geographical location, harvest time, and extraction method. The results of preliminary phytochemical screening of Noni fruits obtained from three different locations were presented in Table 1. It showed that several phytochemical constituents such as alkaloids, flavonoids, quinones, and saponins were found in all extracts from different locations. However, terpenoids and saponins were not presented in the extracts from three locations. The presence of wide range of bioactive compounds in aqueous and alcoholic extracts were accordance with earlier studies (Ranvir et al. 2017).

Furthermore, the total phenol (TPC) and flavonoid content (TFC) in the extracts were also determined using colorimetric method. The results were interpreted as gallic acid equivalent (GAE) and quercetin equivalent (QE) in mg/g extract. As shown in Table 1, Noni fruit extract from Bogor contains the highest values of total phenol and flavonoids compared to others extract with TPC and TFC values of 3.08±0.72 mg/g GAE and 1.11±0.35 mg/g QE, respectively. The TPC value of these three Noni extracts is higher than the TPC value of Noni fruit extract from the Semarang area which is around 0.051±1.677 mg gallic acid equivalent per g of extract (Wigati et al. 2017).

Physicochemical standard gives important information for further investigation and facilitate the identification of formulations in routine industrial production. The results of physicochemical analysis of Noni fruit crude drugs were displayed in Table 2. The result of loss on drying revealed that all crude drugs obtained from different locations were less that10%. It has been known that the value of loss on drying (LOD) at 110 °C should be less than 10%, to eliminate of contamination by mold and growth. Furthermore, determination of total ash content play role in order to evaluate the purity and presence of inorganic material such as sulphated ash, water soluble ash, acid insoluble ash, salt and/or silica in the samples. The results revealed that the total ash and the acid insoluble ash contents were less than 1% for crude drugs and 15% for extracts. Next, the water content refers to the minimum range amount of water content in the extract was determined by toluene distillation. It has been known that water content is associated with quality of extract in which less water content related with less possibility mold contamination. Water content of crude drugs and extracts were between 2.14–3.20%. This result met the standard of PHI (less than 10%). All those parameters meet the requirements of the Indonesian herbal pharmacopeia.

Contamination by heavy metals such as mercury (Hg), copper (Cu), cadmium (Cd), and arsenic (As) in herbal remedies can be attributed with several aspects, including environmental pollution, and can pose clinically relevant dangers for the health of the user and should therefore be limited (WHO 1998). Inductively coupled plasma - optical emission spectrometry (ICP-OES) was used to determined contamination of crude drugs and extracts from heavy metal. As shown in Table 2, there was no heavy metal detected in crude drugs and extracts obtained from three different locations. The results were in agreement to previous study where the maximum limit for Pb ≤ 10 mg/kg, Cd ≤ 0.3 mg/kg, Hg ≤ 0.5 mg/kg, and As ≤ 5 mg/kg (Angelina et al. 2021). The next parameter of herbal quality control is microbial contamination. The result showed that there was no contamination from any bacteria i.e., Escherichia coli, Pseudomonas aeruginosa, Salmonella sp, and Shigella sp., found in all crude drugs and extracts (Table 2). Thus, the results of heavy metal and microbial contamination analysis was in agreement with Indonesian Herbal Pharmacopeia (BPOM RI 2010).

HPTLC has been used as analytical instrument in herbal medicine quality control and fingerprint of plant drugs. Quality control of crude drugs and is based on plant characteristics and morphology and the analysis of marker compounds. It has been reported that the main secondary metabolites of noni fruit are anthraquinones, scopoletin, quercetin, and ursolic acid, as well as some flavonoid compounds (Almeida 2019). According to Indonesian herbal monograph, scopoletin has been used as a biomarker for the standardization of noni fruit extract (PHI 2008). Scopoletin is a coumarin compound which shows a unique spot-on TLC plate under ultraviolet irradiation at of 365 nm which emits blue fluorescence (Fig. 2). Furthermore, the optimum eluent system to achieve TLC profile results showed spots at Rf 0.33. Was hexane: ethyl acetate (2:3). A shown in Fig. 2, scopoletin presence in all Noni extracts from different location is in accordance to standard marker. Based on this result, all those Noni crude drugs and extracts met the requirements of Indonesian Herbal Pharmacopeia. The scopoletin content in crude drugs and extract were 0.02% and 0.38%, respectively. However, only crude and Noni fruit extract from Bogor has scopoletin content higher than the levels that are stated in the monograph, were 0.03% and 0.51%, respectively. It has been known that, one of key factor that affected the quality of herbal medicine in term of its active constituents is location. Differences in growing location and plant age generally cause significant differences in primary and secondary metabolites (Yang et al. 2018), including the marker compound scopoletin in three Noni fruit extracts.

Figure 2. 

HPTLC and TLC chromatogram of standard at various concentration (10, 50, 100, 200, and 500 µg/mL) and samples obtained from different locations (B:Bogor, T:Tangerang, S:Sukabumi).

Almost 200 phytochemicals were reported until 2012 from different parts of Noni fruit (Singh 2012); however, the complete chemical constituent has not been fully reported. Some of the known phytochemicals detected in Noni fruit was scopoletin (Mahanthesh et al. 2013), deacetyl asperulosidic acid and asperulosidic acid (Yang et al. 2009) and rutin (Lewis Lujan et al. 2014). Analysis of three standardized crude extracts using LC-HRMS revealed the presence of scopoletin, which was compared to the authentic standard of scopoletin (m/z = 193.0527 [M+H]⁺, t_R = 3.87 min) shown in Fig. 3. Extracted ion chromatogram of rutin ([M+H]⁺ = 611.1606) on three samples showed a peak (t_R = 3.67 min) tentatively assigned as a rutin. This assignment made on the basis of MS fragmentation pattern which were showed fragmentation at m/z = 463.1784 [M+H]⁺, m/z = 487.2463 [M+Na]⁺ and m/z = 303.0509 [M+H]⁺, similar to observation from Keki et al (2001). In the other hand, extracted ion chromatogram for asperulosidic acid (m/z = 433.1340 [M+H]⁺), deacetyl asperulosidic acid (m/z = 391.1234 [M+H]⁺) and morindin (m/z = 565.1551 [M+H]⁺) showed no presence for those compounds in the standardized crude extract (Table 3). The standardized Noni crude extracts obtained from Tangerang, Bogor, and Sukabumi showed no difference in the BPC chromatograms profile. The relative intensity Fig. 4, showed the highest scopoletin content was present in extract obtained from Bogor (563.050; 6.40 % area) compared to Tangerang (306.956; 3.4 % area) and Sukabumi (441.291; 5.29 % area), which was corroborated the result from the HPTLC analysis.

Figure 3. 

Base peak chromatogram of blanko, and Morinda citrifolia fruit extract obtained from different location. Scopoletin and rutin indicated by the blue arrow.

Figure 4. 

Mass chromatogram of authentic scopoletin (A) and scopoletin presence in Morinda citrifolia fruit extract obtained from Bogor (B). Mass fragmentation of rutin (C) (tentatively assigned).

Noni has been reported to possess several pharmacological properties, including decreasing high blood pressure, lowering blood sugar, and antioxidants. (ACE) plays important role in the renin-angiotensin system to controls blood pressure. ACE catalyzes the conversion of inactive decapeptide angiotensin I to active octapeptide angiotensin II. The use of ACE inhibitors is well established as one of the therapeutic principles in the treatment of hypertension (Serra et al. 2005). In Indonesia, Noni has long been used as herbal medicine to help lower blood pressure; as reported by Yanti et al. 2020, Noni is used with other herbal mixtures as a traditional antihypertensive medicine. Therefore, the present study was also performed to evaluate the pharmacological effect of Noni fruit extract on ACE. In this study, the ACE inhibition assay was carried out using a colorimetric method using hippuryl-glycyl-glycyne (Hip-Gly-Gly) as a substrate and TNBS as a color indicator which will form the yellow color TNP-Gly-Gly, detected at 415 nm (Serra et al. 2005). As presented in Table 4, showed Noni fruit extract from Bogor exhibits strong activity in inhibiting ACE with an IC₅₀ value of 206.26 µg/mL, when compared to extracts from Tangerang and Sukabumi with IC₅₀ value of 232.95 and 220.91 µg/mL, respectively. This finding was supported by a previous study in which the IC₅₀ value of antihypertensive herbal extracts containing noni was around >200 µg/mL (Yanti et al. 2020). In addition, the ethanolic extract of M. citrifolia leaves and fruit also displayed a significant antihypertensive activity in dexamethasone-induced hypertensive rats (Wigati et al. 2017).