Pharmacological Activities, Isolated Compounds, Toxicity, and Potential for New Drug Discovery from the Genus Leea

The genus Leea is widely recognized as a valuable source of medicinal plants, and half of its species are traditionally used to treat various diseases. According to previous studies, it also has significant potential for yielding molecules that can serve as drugs or lead compounds in the discovery of pharmaceutical products. Therefore, this review aimed to identify research gaps in the development of plant-based medicines from the genus Leea. The procedures comprised extracting data from 108 articles, which explored plants from the genus Leea and were published from 1997 to 2022. The articles reviewed consisted of data on pharmacological activity, isolated compounds, and toxicity potential. The results showed that among the 36 species in the genus, L. indica, L. macrophylla, L. asiatica, L. aequata, L. aculeata, L. guinensis, and L. rubra were often used in traditional medicine, leading to their frequent exploration in previous reports . A total of 66 compounds had been isolated, with 46 being known to have pharmacological activity. In addition, the dominant pharmacological activities of these compounds included antioxidant, antimicrobial, anti-inflammatory, and anticancer properties. The acute toxicity test results showed that the extract of plants from the genus was often categorized as not toxic at a dose of up to 2 g/kg BW. Based on the various pharmacological activities of the isolates and extracts, as well as the low toxicity potential, the genus Leea has the potential to be explored for the development of new drugs.


INTRODUCTION
History proves that plants have consistently shown their significance as medicinal sources for treating various diseases.Several reports have also shown that the majority of drugs in prevalent use today are derived from either plant isolates or derivative compounds.In addition, the slogan "Back to nature" has inspired the development of various herbal medicines.Various plant genera, including Leea, have also become the focus of recent studies aimed at discovering and developing new drugs.
Leea is one of 14 genera in the Vitaceae family, which is widely known as the medicinal plant genus.Several studies have reported that the genus consists of plant species widely used in traditional medicine.Various countries, including India, Bangladesh, and Madagascar are known to use these species for the treatment of different diseases.The significance of the genus Leea is evident through its position as the focus of different literature reviews, with 2 being published in 2021.In addition, studies in Bangladesh have extensively explored its traditional uses and pharmacological activities (Hossain et al., 2021).A review of articles on the genus was also simultaneously published in India on the distribution, phytochemistry, and pharmacological activity, specifically for species commonly used in the country (Nehru et al., 2021).This current literature review was carried out to complement and extend the insights provided by these studies.The results are expected to present data on all species of the genus Leea, delving into individual examination of species used in therapy, as well as their pharmacological activity, isolated compounds, and toxicity data.The comprehensive display of isolated compounds and their pharmacological activities are essential in the discovery of new drugs.The toxicity data is also an essential factor, as both single-molecule drugs and 2 Volume 35 Issue 1 (2024) herbal medicines, must fulfill the aspects of efficacy (pharmacological activity) and safety as proven through toxicity tests.
Plants of the genus Leea are typically found growing in the wild without requiring special cultivation need, but the increasing human land usage can cause extinction.Therefore, this review article aims to increase human awareness regarding the crucial need to conserve this genus as a source of new medicine discoveries.The results can also serve as a guide in developing further related studies, particularly those aimed at finding new single-compound drugs and herbal products.

METHODOLOGY
The review began with the tracing of data on all species of the genus Leea on www.theplantlist.orgwebsite.A literature search was then carried out for each species in the database on Science Direct, PubMed, and Google Scholar.Subsequently, all articles collected were screened through their titles and abstracts.The reviewed articles presented data on activity tests, compound isolation, pharmacological activity of the isolated compounds, and toxicity tests of plants from the genus Leea.

Species and Pharmacological Activities
The Plant List website (www.theplantlist.org)stated that there were 199 species of the genus Leea.These names consisted of the accepted names and synonyms, but only 36 of them had been declared taken (Table I).In addition, 14 of these species had been published for medicinal purposes.L. indica and L. macrophylla were the most studied species, followed by L. asiatica, L. aequata, L. aculeata, L. guinensis, and L. rubra (Table I).

Leea indica (Burm F.) Merr
L. indica was a favorite species that was widely studied for the treatment of various diseases.India and Bangladesh were reported to be the leading countries in terms of publications related to this plant species, followed by Malaysia and Singapore.Furthermore, this data was based on the number of articles found through online searches on the Google Scholar, PubMed, and Science Direct databases.Among the 16 publications reviewed in this section, 7, 4, 3, and 2 originated from India, Bangladesh, Malaysia, and Singapore, respectively.
Plant was often used in the traditional medicine of the people of Malaysia, India, Thailand, and China.The roots and leaves were traditionally used in the treatment of cancer, diabetes, diarrhea, dysentery, spasms, and various skin problems (Reddy et al., 2012).Several reports had shown that it possessed antioxidant and cytotoxic properties (Ghagane et al., 2017) (Rahman et al., 2013) and played a role in inhibiting the growth of various cancer cell lines (Wong et al., 2012) and inducing mitochondria-mediated apoptosis in cervical cancer (Wong & Abdul Kadir, 2012).Other studies also showed that it had antiproliferative (Siew et al., 2019), thrombolytic (Rahman, et al., 2013), antimicrobial (Rahman, et al., 2013), anticancer prostate (Ghagane et al., 2017), sedative (Raihan et al., 2011), and anxiolytic properties (Raihan et al., 2011).
In a study conducted by Hsiung (2011), 2 anticancer compounds were isolated, including mollic acid arabinoside and mollic acid xyloside, which belonged to the triterpenoid glycosides group.These compounds could inhibit the growth of Ca Ski cervical cancer cells, each with an IC50 value of 19.21 and 33.33 µM.According to previous reports, these compounds had not been previously identified in L. indica or any other species (Wong et al., 2012).In 2008, a total of 23 compounds were isolated from the plant (Table II).

Leea macrophylla Roxb. ex Hornem
Lee macrophylla was an edible wild plant, that was rich in minerals and vitamins (B1, B2, B12, and C), and could easily be found in South and Southeast Asian Regions, including India, Nepal, Bangladesh, Bhutan, Myanmar, Thailand, Cambodia, and Laos (Joshi et al., 2016).The plant's root had been the focus of several extensive studies, and it was often used in traditional medicine for the treatment of various conditions, such as goiter, colon cancer, lipoma, and tetanus (Mawa et al., 2019).Traditionally, the plant was considered effective in treating guinea worm and ringworm, as well as for healing wounds (Joshi et al., 2016).
This species was not only used empirically in therapy but had also been studied scientifically in several reports.Methanol extract of L. macrophylla roots had also been proven to have analgesic and anti-inflammatory activities.Doses of 100 and 200 mg/kg BW of this extract inhibited the formation of edema in carrageenan-induced animal models.
The antioxidant activity of this plant had also been proven by several studies.According to Islam, extracts from various L. macrophylla seeds exhibited significant antioxidant properties by scavenging free radicals, such as DPPH, superoxide, and NO.This activity could be attributed to compounds, such as oleanolic acid and its derivatives, as well as stigmasterol, which played essential roles in these effects (Islam et al., 2013).Further studies proved that the seed extract had hepatoprotective (Akhter et al., 2015) and neuroprotective activities (Ferdousy et al., 2017), as well as antidiabetic through its protective effect on pancreatic β cells (Mawa et al., 2019).
The methanol extract of L. macrophylla leaves was shown to have the ability to repair damage to the liver tissue of albino Wistar rats induced using CCl4.In addition, CCl4-induced mice showed increased serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP).Treatment with the plant's ethanol extract caused a decrease in the serum levels of these 3 enzymes (Akhter et al., 2015).This study carried out a series of field tests, hole cross tests, EPM, and thiopental sodiuminduced tests.Albino Wistar rats were used in the trial, and open field and hole cross-tests were carried out to assess the locomotor activity of the animals, while EPM was used to evaluate anxiolytic effects.A decrease in locomotor activity indicated a sedative effect (Ferdousy et al., 2017), and the results showed that the extract had central nervous system depressant, anxiolytic, and sedative activity (Ferdousy et al., 2017).Mawa (2019) proved that L. macrophylla root extract could stimulate the performance of the pancreas.Test animals treated with this plant root extract for 3 weeks showed improvement in pancreatic β cells.The results of this study were an early indication of its use as a functional food source for people with type 2 diabetes mellitus (Mawa et al., 2019).

Leea aequata L.
Compared to L. macrophylla, L. aequata was widely distributed in mainland south and southeast Asia.This shrub was one of the plants used in traditional medicine in Myanmar, and its fresh leaves were typically ground for wound treatment (Tun et al., 2019).In addition, the people of Tanah Karo, Indonesia, used them for wound care and as muscle relaxants for tetanus sufferers (Ginting et al., 2018).The seeds, roots, and bark had also showed antimicrobial activity (Tun et al., 2019) (Kujur, 2010).
In a previous study, 23 compounds were successfully isolated from the ethanol extract of the plant's aerial part.Among these, 3,4,5trihydroxybenzoic acid ethyl ester showed antibacterial activity (Tun et al., 2019).Furthermore, its leaves were found to possess antioxidant and antiproliferative properties (Hossain et al., 2021), and this finding was consistent with further reports.A total of compounds from L. aequata leaves, namely 7-Omethylmearnsitrin and roseoside A, had inhibitory effects on HeLa cell proliferation, showing their potential as anticancer agents (Rahim et al., 2021).

Leea asiatica (L.) Ridsdale
The fruit of the L. asiatica plant was usually consumed by people in the northwest Himalayas, India (Singh et al., 2015), and was used to treat various diseases.The Karnataka tribe used this plant for the treatment of fractures, while inhabitants of the Andaman Islands used the roots to treat boils and wounds.Several studies showed that the Tripura tribe used the leaves to treat worm infections and liver diseases.This plant had also been used in several other areas for eye pain, diabetes, and gastrointestinal disorders (Sen et al., 2013).
The usage of this plant for medicine had motivated experts to study and confirm their ideas.The methanol extract of L. asiatica leaves was found to be effective against worm infections and exhibited antioxidant properties (Sen et al., 2012).Furthermore, it also had neuroprotective activity (Sen et al., 2013), hepatoprotective (Sen et al., 2014), anti-inflammatory, and accelerated wound healing.Compared to the leaves, the fruit also had good potential to be studied in more depth due to its rich polyphenol content, which could effectively capture free radicals.The ability of various compounds to inhibit enzyme activity, which led to skin aging and skin darkening, made the fruit worthy of consideration as an ingredient in making cosmetics (Singh et al., 2015).

Leea rubra
Leea rubra was known as the red tree shrub, which could easily be found wild in forests on the continents of Asia and Australia (Das et al., 2022).The Lanna indigenous people in Thailand had traditionally used the roots and bark to treat gastrointestinal diseases (Kadchumsang et al., 2014), while it was commonly used to treat hypertension in Brazil (Braga et al., 2007).
Scientific evidence efforts showed that L. rubra had several pharmacological activities, including antioxidant, anticancer, and antibacterial (Das et al., 2021) (Kadchumsang et al., 2014).The results of other studies showed that this plant also had the potential to be developed as a functional food source.This was primarily attributed to the content of essential amino acids and minerals necessary for health (Awotedu et al., 2018) (Ajiboye et al., 2014).

Leea aculeata
Leea aculeata was often used in traditional medicine and was easy to find in low to moderate plains in forests along rivers.Furthermore, it was a shrub plant (Gonzales et al., 2019), which had traditionally been used as an antipyretic, postpartum care, poultice, and for the treatment of headaches (Villazorda, 2015).Scientific studies proved that it had antioxidant (Villazorda, 2015) and anti-hyperuricemia activity by inhibiting xanthine oxidase enzyme activity (Gonzales et al., 2019).

Leea alata
Leea alata was a medicinal plant, and its root was commonly used in the treatment of jaundice by the indigenous people of Korku, India (Choudhary and Upadhyaym, 2012).

Leea thorelii
Leea thorelii was a small shrub that grew wild and had traditionally been used by Thai people as an antipyretic and anti-inflammatory agent (Kaewkrud et al., 2007).Kaewkrud et al. (2007) had successfully isolated 5 compounds from its leaves, such as citroside, a megastigmane that had not been reported from any other species, while the structure of the other 4 compounds had not yet been determined (Kaewkrud et al., 2007).Lakornwong et al. (2014) successfully isolated 8 compounds from its roots, including bergenin and 2 of its derivatives, a gallic acid derivative, 3 flavan compounds, and a coumarin (Table II) (Lakornwong et al., 2014).

Leea angulata
The people of the Sasak tribe in Lombok, Indonesia, usually used the bark of L. angulata plant for wound healing.The community stated that the use of its bark could reduce pain and improve wound healing (Hidayah & Barlian, 2022).Furthermore, Hidayah et al. (2021) proved that the plant bark extract could accelerate the proliferation process, thereby accelerating wound healing (Hidayah & Barlian, 2022).

Leea guineense
Leea guineense was traditionally used as an anti-inflammatory agent due to its composition of essential compounds (De Beck et al., 2003).De Beck et al. 1999 successfully isolated a flavonoid, namely quercitrin-3'-sulphate from the plant, while 2 other quercitrin sulphate compounds were identified in 2003 and their antioxidant activity was tested.In addition, the 2 new compounds were quercitrin 3,3'-disulphate and quercitrin 3,3',4'trisulphate (De Beck et al., 2003).Studies on this plant ended in 2003, as evidenced by the absence of publications regarding its potential as a medicinal plant.

Leea philipinensis
Leea philipinensis was an endemic plant in the Philippines, and there were no reports on its empirical use for treatment.However, a previous study proved this plant had antioxidant activity due to its beneficial composition (Santiago & Bartolome, 2015).

Leea tetramera
Based on previous reports, there were no publications on the use of Leea tetramera in traditional medicine.Khan et al. proved that the plant extract could inhibit the growth of various bacteria (Khan et al., 2003), but after 2003, there were no more publications on its potential for treatment.

Isolated Compounds
Several species of this genus had provided an increased vocabulary of potential chemical compounds to be developed as drugs, food additives, and others.L. indica, L. macrophylla, L. asiatica, and L. aequata were the prototype plants of this genus, which had been intensely studied for the isolation of constituent compounds.Several isolates of these compounds had also been tested for their activities (Table II).

Toxicity
Toxicity tests of plants from the genus Leea were generally still limited to acute oral toxicity tests and were often declared safe.Based on findings, no death cases had been reported during acute toxicity test, and observations of vital organs (heart, liver, kidneys, lungs, and brain) showed no toxicity signs (Table III).

CONCLUSION
In conclusion, L. macrophylla, L. indica, and L. aequata were species of the genus Leea, which had been extensively explored.These plants were used in traditional medicine and had been tested for their activity, toxicity, and phytochemical content.In addition, their acute oral toxicity test showed a low toxicity potential, while the activity test showed promising pharmacological properties.Based on these findings, the plants had the potential to be developed into medicine.Phytochemistry study also found that some compounds had pharmacological activity as a single compound, and could be developed into new drugs or lead compounds to discover new drug molecules.No deaths were up to a dose of 2 g/Kg BW.In subacute toxicity, no signs of toxicity were found.There were no significant changes in body weight and the kidneys, liver, heart, and brain weight.These organs also did not show any histological changes.Biochemical and hematologic parameters were almost the same as control animals (Akhter et al., 2015) Root tuber ethanol extract The safety limit of the extract is 5 g/Kg BW when administered orally.The section did not cause behavioral modulation, symptoms of toxicity, and morbidity.In acute dermal toxicity: the extract does not induce swelling, inflammation, irritation, or other skin abnormalities (Joshi et al., 2016)

L. rubra
One of the isolates, myricetin 4'-methoxy-3-Oα-Lrhamnopyranosid In the research procedure, it was mentioned that there was a determination of LD50 in experimental rats, but the results section did not find this data (S.Das et al., 2022)

Leaves ethanolic extract
There were no deaths after administration of a single dose up to 2000 mg/Kg BW (Bulbul et al., 2022)

Other species
No toxicity test data found International Jornal of Bioassays, 3 (7) Table II Continue.