A Study Uncover Itchy Leaves Ethnomedicine Usage: A Preliminary Study on Characterization and Bioactivity of Laportea Spp

Background: Laportea decumana (Roxb.) Wedd. and Laportea stimulans (L.f.) Miq. (Urticaceae) or itchy leaves are endogenous plants from The Maluku Islands, Indonesia, which are used topically as analgesics by local people. Objective: As part of a preliminary investigation on the itchy leaves, this study was conducted to provide information on their macroscopic and microscopic characteristics, phytochemical profiles, and bioactivities according to the ethnomedicinal usages. Materials and Methods: The macroscopic examination was carried out on fresh leaves. The microscopic examination was carried out on leaf powder under a light microscope. The phytochemical analyses using TLC were conducted on those crude extracts and fractions. The bioactivity assays were conducted in vitro as Cox-2 inhibitory and DPPH radical scavenging activities. Results: reveal similar characteristics in the macroscopic and microscopic properties of both Laportea spp. leaves. Calcium oxalate crystals were observed in L . decumana but not found in L . stimulans . Both species have stomata with anisocytic type. More trichomes are found in the leaves of L. decumana , yet smaller and easily detachable. TLC analyses exhibited slightly different profiles. The crude extracts and fractions at 10 µ g/ml showed similar inhibitory percentages on Cox-2. The DPPH scavenging activities of the crude extracts and fractions of L. decumana showed active moderate activity with an IC 50 value < 250 µ g/ml, while those of L. stimulans showed moderate to weak potency with an IC 50 value < 500 µ g/ml. Conclusion: Further exploration on Laportea spp. pharmacological activity is recommended to provide stronger evidence for its ethnomedicinal usage.


INTRODUCTION
With a vast biodiversity of 20,000 types of plants, 1 Indonesia provides various indigenous medicinal plants used for generations 2-4 based on indigenous knowledge and local wisdom. 5 Despite broader usage by the community, lack of scientific data is a significant issue for traditional medicine development, worldwide. 6 Itchy leaves are plants used traditionally by local people in eastern Indonesia, mainly in the Maluku Islands, to relieve muscle aches following hard work in the field. Records of the ethnobotanical use of itchy leaves are to reduce pain, fatigue, headaches, stomachaches, and muscle pain. 7,8 At least two main species named itchy leaves were identified later as two different species. 8 Laportea decumana and Laportea stimulans 9 belong to family of Urticaceae, which are typical plants that grow in Maluku 10 dan Papua. 7 Laportea spp. is widely distributed in Maluku and Papua. 11 The community cultivates those plants in the house yard and sells the leaves in traditional markets. 10,12 The L. decumana 13 is used more often by the local people since it is relatively easier to be found and more convenient to apply on skin. 10 In an effort to widen the itchy leaves utilization as herbal products, a thorough investigation of the plants' pharmacological effects should be accompanied with a proper quality assurance process. 14 Considering there are plants with similar local name, it is of importance to provide a source identification as the initial stage in determining the bioactive components of a plant and ensuring the correctness, quality, and proper use of the simplicial. 15 This purpose can be achieved through a macroscopic, microscopic, and phytochemical comparison of the leaves of these similar plants, Laportea decumana and Laportea stimulans. 7,9,10,16 This study aims to characterize the leaves of L. decumana and L. stimulants macroscopically and microscopically, as well as to conduct a phytochemical screening of these plants by using Thin Layer Chromatography. A preliminary investigation of the bioactivities was conducted by evaluating their Cox-2 inhibitory assay and DPPH radical scavenging activity by in vitro methods.

Macroscopic characterization
Macroscopic examination was carried out by observing the morphology and size of the fresh leaves and carrying out organoleptic tests including the taste, flavor, and color of the dried powder. 17

Microscopic characterization
Microscopic examination, the observed part includes, the upper epidermis section of the section used is leaf slices from the upper leaf surface measuring 1x1 cm. For examination of leaf powder used powder from the leaves. Prepare a glass object and drop it with 1-2 drops of chloral hydrate, then heat it over a spirit lamp and then cover it with a glass cup. Then observed under a microscope with revolver objective lens of 40 × magnification. 17

Sample preparation
Leaves were collected from village Nuanea districts center of the Maluku Islands, Seram Island, Indonesia on December 2021. Leaves samples were immediately processed to avoid damage of the secondary metabolites contained therein; 18 washed, chopped, and dried. The drying process was done at room temperature protected from direct sunlight. Afterward the dried samples were powdered. 19 Extraction Dried powder was macerated by using 96% ethanol p.a as a solvent in a ratio of 1:10. 19 The macerate was filtered by using a Buchner funnel, followed by evaporation with a rotary vacuum evaporator at a temperature of 60 °C at a speed of 80-110 rpm to obtain a thick extract. 20,21 Fractionation The fractionation process is carried out using the liquid-liquid partition method using a separating funnel. The crude extract was successively partitioned to gain the n-hexane and ethyl acetate fractions. 20,21 Phytochemical screening The screening was carried out semi-quantitatively with thin layer chromatography technique. 20,21 The plate was activated by heating it in an oven at 50 0 C, approximately10 minutes prior sample application. 1 The TLC system for the crude extracts, hexane and ethyl acetate fractions used a precoated silica G 60 GF 254 plate as the stationary phase, while the mobile phase used were as follows n-hexane: ethyl acetate (7:3) v/v. The system used for the water fraction was a precoated silica G 60 GF 254 plate as the stationary phase and the mobile phase used were as follows methanol: ethyl acetate: formic acid (7:3:0,2) v/v.

Detection for flavonoid compound
Extracts from L. decumana and L. stimulans were separately dissolved in methanol and then each was applied 1 mg/mL using Linomat  on to a precoated silica gel plate F254 and eluted using the aforementioned mobile phase. Afterwards, dried plate was sprayed by AlCl 3 reagent. The plate was observed under UV lamps of 254 nm and 366 nm before and after spraying. Quercetin was used as a reference standard.

Detection for phenolic compounds
Extracts from L. decumana and L. stimulans were dissolved methanol with a concentration of 1 mg/mL and then each was applied 3 µL using Linomat  on to a precoated silica gel plate F 254 and eluted using the aforementioned mobile phase. Afterwards, dried plate was sprayed by a FeCl 3 reagent. The plate was observed under UV lamps of 254 nm and 366 nm before and after spraying. Gallic acid was used as a reference standard.

Detection for alkaloids compounds
Extracts from L. decumana and L. stimulans were dissolved methanol with a concentration of 1 mg/mL and then each was applied 3 µL using Linomat  on to a precoated silica gel plate F 254 and eluted using the aforementioned mobile phase. The plate was observed under UV lamps of 254 nm and 366 nm and sprayed by a dragendroff reagent. Quinine was used as a reference standard.

Detection for terpenoids compounds
Extracts from L. decumana and L. stimulans were dissolved in methanol with a concentration of 1 mg/mL and then each was applied 3 µL Linomat onto a precoated silica gel plate F 254 and eluted using the aforementioned mobile phase. Then the plate was dried and sprayed with annisaldehyde H 2 SO 4 and observed under UV lamps at 245 nm and 366 nm. Stigmasterol was used as a reference standard.

Cox-2 inhibitory assay
The assay was carried out in vitro using a Cox-2 (human) inhibitor screening assay kit (Cayman Chemical) in accordance with the standard procedures listed on the kit and was dissolved methanol with a concentration of extract and fraction used 10 µg/ml. 22,23 The assay was done in triplicates. The percentage of inhibition was calculated by the equation as follows: % Inhibition = Abs control -(Abs extract -Abs blanko) x 100% Abs control

DPPH radical scavenging activity
The extracts and fractions were separately dissolved in methanol p.a followed by a serial dilution. To each solution was added 0.1 mM DPPH solution in a ratio of 2:1, followed by homogenization. 24 The solution was then incubated for 30 min in the dark, and the absorbance was measured at a wavelength of 516 nm. 25 The blank solution used methanol p.a. The assay was done in triplicates. The ability to inhibit DPPH radicals was calculated by the equation as follows: % radical scavenging activity = Abs control -Abs sample x 100% Abs control

Statistical analysis
The data obtained from the Cox-2 inhibitory and the DPPH radical scavenging activity were analyzed statistically by one-way ANOVA or Kruskal-Walli's test with 95% significance level, which were processed by SPSS 24.

Macroscopic characterization
The macroscopic observation was conducted to differentiate the morphology, size, colour of the leaves of L. decumana and L. stimulans.
Prior to the macroscopic examination, organoleptic tests were carried out, including taste, flavour and colour (Table 1).    L. decumana leaf is a green heart-shaped which tip is pointed, and the leaf edge is serrated and oval. It has trichomes on the leaf surface, behind the leaves and bones, and the petiole is red and has a length of 24.5 cm and a width of 14.7 cm. L. stimulans leaf color is green, oval shape, pointed tip, and elliptical leaf edge. There are trichomes on the surface of the leaves, behind the leaves, and the bones and stalks. The leave has a length of 32 cm and a width of 15 cm (Figure 1).
L. decumana leaf has a light green color on its upperside, while on the other side is purplish red. L. stimulans leaf has a dark green upperside, and the other side of the leaves was green. L. decumana has an oval leaf shape and tapered leaf tips in all parts of the leaf. There are many tight trichomes. While L. stimulans have an oval leaf shape and pointed leaf tip, the shape is larger, and the trichomes are rare but firm on the upperside and and the other side of the leaves.

Microscopic characterization
Result of the microscopic observation of L. decumana fresh leaf as described on Figure 2, exhibited a phloem and xylem ladder form, secretory cells located on the upper epidermis and there are 3-7 palisade in the upper epidermis ( Figure 2).
Result of the microscopic observation of L. stimulans fresh leaf as described on Figure 3, exhibited carrier bundles as ladder-shaped, secretory cells located on the upper epidermis and stomata of an anisocytic type.

Phytochemical screening
Phytochemical screening exhibits similar TLC profiles of both crude extracts having similar Rx for the spots reacted to the flavonoids, phenolics and terpenoids reagents sprayed while exhibited different Rx for the spots reacted to AlCl 3 and FeCl 3 ( Table 2 and 3). Both crude extracts showed similar Rx and colour change following the annisaldehyde H 2 SO 4 spraying ( Table 2, 3 and 4). DPPH spraying showed similar positive spots of both Laportea however those of L. decumana exhibited additional positive spots. Table 4 summarize the results while also exhibited the positive result of the alkaloids detection by using dragendorff as the spraying agent.

DISCUSSION
Research on medicinal plants and herbs ("Ristoja") in 2017 reported Indonesia biodiversity covers 11,218 medicinal plants, of which 9,516 plant species used as part of traditional remedy. 26 10 to overcome various health complaints. 8 Itchy leaves are used topically to relieve aches, fatigue, headaches, stomach aches, joint and muscle aches, and bruises. 7 The local people usually rub the leaves on the painful area, and afterwards there will be a stinging sensation followed by numb and anesthetic sensation. 8 L. decumana widely grows and well distributed in the Maluku Islands and Papua. It can be found in humid areas, and most of these plants grow side by side with Sago palms, next to a small river. 11 The plant is also cultivated in the houseyard, and easily found also in the traditional markets. 10,12 On the other hand, the L. stimulans is also named by the local people as itchy leaves, while it also can cause itching when is applied on skin.  +  71  -+  ----n-Hexane fraction  12  -+  ----+  18  -+  -----27  +  +  -----37 -   Dark yellow  ----53  -+  -----60  +  +  Dark yellow  Dark blue  -Brick red  +  70  -+  -----80 -     Note: Statistical analyses on L. decumana showed significant difference between the extract and the fractions at various concentrations following the DPPH radical scavenging assay with a value 0.00 <0.05. It grows well in the Maluku area but not as much as L. decumana. It is rarely used topically since it induces pain following application.
The leaves of L. decumana and L. stimulans can be easily differentiated macroscopically (Figure 1). On the other hand, the major difference of the microscopic characteristics was observed on the absent of oxalate crystals in L. stimulans.
Trichomes of the family of Urticacae easily fall off by touch; and have a sharp shape like syringes. 27 Syringes can penetrate the skin and release irritants. 28 In L. decumana the presence of more dense and dense trichomes are located on the leaf surface but easily detached, biomineralized, and less sharp in contrast to L. stimulans trichomes, which are rare on the leaf surface but have a more muscular shape and are attached to the leaf base which can penetrate the skin and provide more pungent irritant. 29,30 The trichomes in L. decumana are easy to be detached. When the trichomes are separated from the base of the leaves, the injured plant cells cause the cells to secrete formic acid compounds, which provide an analgesic effect. 7 When L. decumana is applied to the skin surface, formic acid secretion occurs from the leaf trichomes, which will cause the skin pores to widen and further relieves aches, pains, and fatigue in the muscles and body. 7 The exact mechanism is not fully understood. At the same time, the trichomes of L. stimulans are fewer but larger in size to induce more dominant pain.
The phytochemical screening of both crude extracts detected the presence of flavonoids, phenolics, alkaloids, and terpenoids. Both leaves seem to have a similar chemical content based on the TLC profile focusing on phytochemical groups characterization; however different TLC profiles were observed amongst crude extracts and the respective fractions following the DPPH spraying. This finding is consistent with the results of the DPPH radical scavenging activity, showing that the L. decumana crude extracts and fractions have higher activity in comparison to those of L. stimulans.
The crude extracts and the fractions from both leaves exhibited similar Cox-2 inhibition assayed on 10 µg/ml (Table 5). Several phytochemicals have been reported elsewhere to exhibit analgesic and anti-inflammatory activities by inhibiting Cox enzymes 31,32 of which some refers to terpenoids, phenolics, and flavonoids as the responsible bioactive compounds. 33,34 Plant extract might serve as a pain reliever through a mechanism by inhibiting the Cox-2 enzyme. 31 By inhibiting the Cox-2 enzyme, prostaglandins as the pain mediators are not formed, thereby reducing pain. [35][36][37] However, the extract and fractions exhibited significant difference in the DPPH radical scavenging potency. 38 It is interesting to explore further whether or the radical scavenging activity correlated to the Cox-2 inhibitory mechanism and further exerts the analgesic action of the leaves. 31,32 This measurement of the DPPH radical scavenging activity was carried out using the visible spectrophotometric method. 25,39 This method was selected due to the prediction of the possibility of the contribution of flavonoid and phenolic contents to the antioxidant activity. Those group of compounds is widely reported to contribute to the DPPH radical scavenging activity which is further responsible for anti-inflammatory activity. 25,39 Reactive oxygen species are caused by oxidative stress conditions, cellular damage occurs and causes various inflammatory diseases, through the activity of membrane phospholipase which catalyzes the biotransformation of arachidonic acid to prostaglandins and thromboxanes by the cyclooxygenase enzyme activity. 40 Therefore, much attention has been paid to correlating the radical scavenging activity of plant extracts as an initial screening for an anti-inflammatory and analgesic activity. 41 The radical scavenging activity was indicated by a decrease in the absorbance of the DPPH solution to which the sample had been added. [42][43][44][45] Differences in the IC 50 values of extracts and fractions of both leaves are in accordance with the difference in the TLC profiles following the DPPH spraying, which confirmed different chemical contents of both leaves. 46,47 The DPPH radical scavenging activity is classified into 5 groups, i.e., highly active (<50 µg/ml), active (50-100 µg/ml), moderate (101-250 µg/ml), weak (250-500 µg/ml) and inactive (>500 µg/ml). 48 Based on the aforementioned classification, only the water fraction of L. decumana showed an active for radical scavenging activity, while crude extract, hexane and ethyl acetate fraction of L. decumana and the water fraction of L. stimulans exhibited moderate activity, while others are classified as inactive or weak. 48,49 Noteworthy, the water fraction of both Laportea showed higher DPPH radical scavenging activity in comparison to its crude extract, hexane and ethyl acetate fractions. A similar TLC profile was also observed but with a slight difference in polarity. Positive reaction to AlCl 3 , FeCl 3 suggests the presence of a flavonol derivate, while the high polarity property as well as the positive reaction to the annisaldehyde H 2 SO 4 spraying suggesting the glycoside form. Further structure elucidation following isolation of the respective compounds is needed to ensure the result.
The L. decumana extract showed a moderate DPPH radical scavenging activity with an IC 50 value of 146.28 µg/ml; the n-hexane fraction had a moderate antioxidant activity with an IC 50 value of 207.86 g/ml; the ethyl acetate fraction had a moderate antioxidant activity with an IC 50 value of 170.75µg/ml, and the water fraction has weak antioxidant activity with an IC 50 value of 66.15 µg/ml (Table 6). On the other hand, the L. stimulans extract showed a weak DPPH radical scavenging activity with an IC 50 value of 522.45 µg/ml; the n-hexane fraction had weak antioxidant activity with an IC 50 value of 453.94 µg/ml; the ethyl acetate fraction had weak antioxidant activity with an IC 50 value of 469.95 µg/ml, and the water fraction has weak antioxidant activity with an IC 50 value of 105.55 µg/ml ( Table 7). The radical scavenging activity is probably related to the presence of phenolic and flavonoid compounds and their structural types. 50 The fact that the crude extract and fractions are L. decumana are consistently exhibited a radical scavenging activity, supports the preference of its usage by the local community, besides its more convenient usage. Itchy leaves, i.e., L. decumana and L. stimulans are both used topically as analgesic by the local people in the Mollucas and Papua. However, there are also other types of itchy leaves plants reported from the same ordo. i.e., Laportea bulbifera, Laportea interrupta. [50][51][52] A further study on the potency of the itchy leaves extracts according to the ethnomedicinal usage are necessary to be conducted to support its development as a standardized and scientifically proofed herbal medicinal products.

CONCLUSION
Itchy leaves of L. decumana and L. stimulans can be differentiated easily according to its appearance in color, shapes of fresh leaves, as well as the typical trichomes and the absent of the oxalate crystals in L. stimulans. Despite having similar COX-2 inhibition activity, L. decumana crude extract and fractions showed higher potency as the DPPH radical scavenging activity in comparison to L. stimulans. TLC profiles suggesting different chemical contents which responsible for the pharmacological activity. It is noteworthy to further investigate the active ingredients of the leaves as well as to provide pharmacological evidence by an in vivo assay to support the ethnomedicinal usage.

ACKNOWLDEGMENT
Authors gratefully acknowledge the research funding from the Faculty of Pharmacy, Universitas Gadjah Mada in 2022.