Structure-Activity-Relationship and Mechanistic Insights for Anti-HIV Natural Products

Acquired Immunodeficiency Syndrome (AIDS), which chiefly originatesfroma retrovirus named Human Immunodeficiency Virus (HIV), has impacted about 70 million people worldwide. Even though several advances have been made in the field of antiretroviral combination therapy, HIV is still responsible for a considerable number of deaths in Africa. The current antiretroviral therapies have achieved success in providing instant HIV suppression but with countless undesirable adverse effects. Presently, the biodiversity of the plant kingdom is being explored by several researchers for the discovery of potent anti-HIV drugs with different mechanisms of action. The primary challenge is to afford a treatment that is free from any sort of risk of drug resistance and serious side effects. Hence, there is a strong demand to evaluate drugs derived from plants as well as their derivatives. Several plants, such as Andrographis paniculata, Dioscorea bulbifera, Aegle marmelos, Wistaria floribunda, Lindera chunii, Xanthoceras sorbifolia and others have displayed significant anti-HIV activity. Here, weattempt to summarize the main results, which focus on the structures of most potent plant-based natural products having anti-HIV activity along with their mechanisms of action and IC50 values, structure-activity-relationships and important key findings.


Introduction
Acquired immunodeficiency syndrome (AIDS) is a disease of the cell-mediated immune system or T-lymphocytes of the human body. In AIDS, the count of helper T cells isreduced, which directly stimulates the production of antibodies from B-cells. Consequently, the body's natural defense system against AIDS infection isdestroyed [1]. According tothe World Health Organization (WHO), it is estimated that about 75 million individuals have been infected from the human immunodeficiency virus (HIV), and about 37 million people are still under the fighting stage. The prevalence of HIV is expected to increase significantly due to illiteracy, non-hygienic living conditions, unsafe sexual relationships and lack of awareness [2]. Initially, the first human retrovirus was founded at the National Cancer Institute, in the USA by Robert Galloand his colleagues, after being first discovered in 1981

The HIV Structure
The motor agent for AIDS is an animal retrovirus named HIV that is ready to replicate and integrate its infectious DNA into the host cell's healthy DNA. It is an animal virus that chiefly attacks the body's helper T cells [6,7]. The virus is spherically shaped, having a diameter of around 90-120 nm. Its genetic material generally contains a single standard RNA fiber metameric into two similar fibers and is connected with an enzyme called reverse transcriptase (RT). The viral coating contains a lipid bilayer that is derived from the membrane of the host cells and spikes of glycoprotein that are like projecting knob. It consists of two protein coats, as depicted in Figure 1 [8,9]. Internally, the virus contains a protein layer (the matrix), which consists of the necessary proteins and nuclear material. The virus also contains an enzyme known as a protease that disintegrates the viral polyproteins to form new functional proteins. The role of reverse transcriptase is to catalyse the conversion of the viral RNA into viral DNA and integrase, which allows the entry of viral DNA into the host nucleus [10,11].  [10]. Image was originally published within Open Access license.

Replication Cycle of HIV
The complete HIV replication cycle is represented in Figure 2. After the entrance of the virus into the body of an individual, the virus invades the body cells through CCRS or CXCR4 receptors shown on the top of the macrophages, known as T-lymphocytes, dendritic cells and monocytes [6,7]. After entering the host cell, the virus binds with chemokine receptors and interacts with cell membrane proteins. The virus then releases and utilizes its reverse transcriptase (RT) enzyme for the synthesis of viral DNA from its viral genome i.e., HIV RNA. This conversion allows the virus to enter into the host cell nucleus, where the enzyme integrase releases and perform integration of its viral DNA into the host cell's DNA [8][9][10][11]. Newly formed HIV proteins and viral RNA shifts towards the cell membrane and reuniteswith immature HIV. The new immature (non-infectious) virus then buds off from the host cells, which inturn initiates the release of the protease enzyme from the viruses that cause the breakdown of long-chain polypeptides of immature viruses. The newly formed small protein particles make the new mature viruses that enter into the new host cells for spreading the infection [12,13].  [10]. Image was originally published within Open Access license.

Replication Cycle of HIV
The complete HIV replication cycle is represented in Figure 2. After the entrance of the virus into the body of an individual, the virus invades the body cells through CCRS or CXCR4 receptors shown on the top of the macrophages, known as T-lymphocytes, dendritic cells and monocytes [6,7]. After entering the host cell, the virus binds with chemokine receptors and interacts with cell membrane proteins. The virus then releases and utilizes its reverse transcriptase (RT) enzyme for the synthesis of viral DNA from its viral genome i.e., HIV RNA. This conversion allows the virus to enter into the host cell nucleus, where the enzyme integrase releases and perform integration of its viral DNA into the host cell's DNA [8][9][10][11]. Newly formed HIV proteins and viral RNA shifts towards the cell membrane and reuniteswith immature HIV. The new immature (non-infectious) virus then buds off from the host cells, which inturn initiates the release of the protease enzyme from the viruses that cause the breakdown of long-chain polypeptides of immature viruses. The newly formed small protein particles make the new mature viruses that enter into the new host cells for spreading the infection [12,13]. The HIV replication cycle [13]. Image was originally published within Open Access license.

Diagnosis
The level of HIV infection is diagnosed from the blood plasma of the host through their viral RNA mass estimation. The infection has been associated with the period of acute symptoms viz; lymphadenopathy, fever, weight loss, lethargy, general malaise, pharyngitis, rashes, nausea, headache, myalgias and meningitis, etc. [2,6]. During acute HIV infection, the viral RNA is at the highest levels in the blood plasma. It is estimated that the amount and characteristics of the virus indicate its pathogenesis and replication. Hence, the clinical details and infection progression depend on the host characteristics, along with the viral genotype [14][15][16]. ELISA and Western Blotting were the two main tests employed for the diagnosis of AIDS in the past. ELISA is used for the detection and measurement of the antibodies that are produced against a specific pathogen [17], while Western Blotting was employed for confirmation of ELISA positive tests. It is used to check the specific proteins in the blood sample. The samples go through the protein denaturation and then gel electrophoresis. The combined effect of both tests is found to be 99% accurate. Nowadays, various advantageous alternatives are available in place of Western Blotting.Among the advantages associated with such alternatives is less time-consuming testing [18].

Present Therapy for HIV/AIDS
The knowledge of HIV has beenmade public since the 1980s. However, there is currently no availability of efficacious therapy or vaccine for the entire destruction of the virus [2]. Current AIDS treatments have many drawbacks, e.g., complex and tedious treatment protocols, requiring expertise from medical practitioners, solid motivation and patient's commitment. Antiretroviral therapy (ART) is only about twenty years old, meaning that further approaches are still in progress. The usage of certain medications can slow the progress of the disease without the patient necessarily being promised total recovery. However, with the development of new entities and immune modulators, it is now feasible to fight this deadly disease [19][20][21]. The drugs provide a meaningful advancement in mitigation, control, cure, and prevention. With the establishment of highly active antiretroviral therapy (HAART) and anti-retroviral agents in 1996 decreased the mortality and morbidity of AIDS has been observed. Antiretroviral therapy is presently prescribed for all adult patients living with HIV [2]. Many types of combination approaches such as the use of nucleoside reverse transcriptase inhibitors, fusion inhibitors, non-nucleoside reverse transcriptase inhibitors, Figure 2. The HIV replication cycle [13]. Image was originally published within Open Access license.

Diagnosis
The level of HIV infection is diagnosed from the blood plasma of the host through their viral RNA mass estimation. The infection has been associated with the period of acute symptoms viz; lymphadenopathy, fever, weight loss, lethargy, general malaise, pharyngitis, rashes, nausea, headache, myalgias and meningitis, etc. [2,6]. During acute HIV infection, the viral RNA is at the highest levels in the blood plasma. It is estimated that the amount and characteristics of the virus indicate its pathogenesis and replication. Hence, the clinical details and infection progression depend on the host characteristics, along with the viral genotype [14][15][16]. ELISA and Western Blotting were the two main tests employed for the diagnosis of AIDS in the past. ELISA is used for the detection and measurement of the antibodies that are produced against a specific pathogen [17], while Western Blotting was employed for confirmation of ELISA positive tests. It is used to check the specific proteins in the blood sample. The samples go through the protein denaturation and then gel electrophoresis. The combined effect of both tests is found to be 99% accurate. Nowadays, various advantageous alternatives are available in place of Western Blotting.Among the advantages associated with such alternatives is less time-consuming testing [18].
Taken together, the present review highlights the discovery of plant-based molecules during the last few decades that have been used in the management of HIV. A detailed account of plants according to their mechanism of action and activity of secondary metabolites has been discussed. In addition to the structures of most potent phytochemicals, mechanistic insights revealed during the biological evaluation, IC 50 values and important key findings have also been presented. The detailed mechanisms of this action and structure-activity-relationships of some of the compound classes remain to be further investigated. This assemblage will be of great help for the scientific community working towards the development of anti-HIV drugs. In this review, the natural medicinal plants are described in two categories:

1.
Plants according to their mechanism of action.

2.
Plants according to the activity of secondary metabolites.

Natural Plants According to Their Mechanism of Action
Therapeutic agents of natural origin may be an encouraging alternative solution for the treatment of several disorders and conditions [53][54][55][56][57][58][59]. In anti-HIV research, attention is chiefly paid tocompounds which interfere with several steps involved in the HIV replication process. For example, almost all the anti-HIV drugs act against the viral proteins represented by the viral protease, integrase, and reverse transcriptase [60]. Anti-HIV drugs can be classified into several groups according to their action on the life cycle of HIV [61]. Hence, different drugs act on these different steps of replication and inhibit the further expansion of the virus into the body. A group of researchers reported the activities of HIV-PR inhibitors from different plants primarily divided into the following categories [62][63][64][65][66][67][68][69][70][71] Fusion inhibitors are also known as Entry inhibitors. These are mainly CCR5 co-receptor antagonists which inhibit the binding of HIV surface glycoproteins with the host cell's receptor [72]. Infection primarily starts with the binding of the viral gp120 to the CD4 cell receptor expressed on the surface of T cells, macrophages, and some monocytes. This results in the conformational change which further stimulates the interaction of secondary gp120 with co-receptor CCR5 [73]. FIs prevent the entry of the virus into the host cell by inhibiting the fusion of virus particles with the membrane of the host cell, which is the early first step of virus replication [74].
Phytoconstituents from some plants, like Listeria ovate, Cymbidium hybrid, Hippeastrum hybrid, Epipactis helleborine and Urtica dioica possessing the activities of fusion inhibitors and act against the HIV-1 and HIV-2 [75,76]. Matsuda et al. reported an alkaloid Cepharanthine (1) isolated from Stephania cepharantha having anti-HIV and anti-tumour potential without exerting any type of serious toxic effects. This compound modifies the plasma membrane fluidity and prevents viral cell fusion [77]. A diterpene lactone named Andrographolide (2) shown in Figure 3 was obtained from the herb Andrographis paniculata and possesses HIV-1 fusion inhibition propertiesevaluated in vitro using AZT (Zidovudine) as a positive control [78][79][80][81][82]. Several other derivatives have been derived synthetically to exert more potent anti-HIV properties [83,84]. 6

Plant Extracts as Reverse Transcriptase Inhibitors
The HIV virus utilizes the reverse transcriptase enzyme for the conversion of its viral RNA into DNA. RT inhibitors mainly act upon this enzyme and prohibit one of the essential steps of viral replication [85,86]. Several natural products have been isolated from plants are available in theliterature, which have been screened for their activity against RT [66]. The plants which tested positively for reverse transcriptase inhibition include; Culendula officinalis, Acacia mellifera, Uvaria angolensis, Hypericum scruglii, Spaganium stoloniferum, Calophyllum brasiliense, Maytenus buchanani, Prunus Africana, Vernonia jugalis, Maytenus senegalensis, Melia azedarach, Calophyllum inophyllum, Lomatium suksdorfii, Coriandrum sativum, Chrysanthemum morifolium and Swertia franchetiana [47,. Capryl aldehyde and methyl-n-nonyl ketone obtained from Houttuyniu cordata directly inhibit the RT enzyme [66]. Calanolides A (3) and B (4) [89] have been obtained from the plant Calophyllum inophyllum. The introduction of bulky groups has been shown to be essential at the C-4 position to enhance anti-HIV activity. The stereochemistry of the C-12 hydroxyl (R or S configured) is not, however, as critical for activity. Methyl groups at the C-10 and C-11positions were also shown to be required for activity. Hydrogen bond acceptors at C-12 were also shown to be responsible for the activity, both in calanolides and inophyllums. In vitro assay results revealed that (+)-Calanolide-A inhibits RT in two diverse template primer systems. The action of (+)-Calanolide-A is possible due to the bi-bi prearranged mechanism of RT. Calanolide is at least partially competitive about dNTP binding. Structure-activity-relationships and important key findings of Calanolides are shown in Figure 4.

Plant Extracts as Reverse Transcriptase Inhibitors
The HIV virus utilizes the reverse transcriptase enzyme for the conversion of its viral RNA into DNA. RT inhibitors mainly act upon this enzyme and prohibit one of the essential steps of viral replication [85,86]. Several natural products have been isolated from plants are available in theliterature, which have been screened for their activity against RT [66]. The plants which tested positively for reverse transcriptase inhibition include; Culendula officinalis, Acacia mellifera, Uvaria angolensis, Hypericum scruglii, Spaganium stoloniferum, Calophyllum brasiliense, Maytenus buchanani, Prunus Africana, Vernonia jugalis, Maytenus senegalensis, Melia azedarach, Calophyllum inophyllum, Lomatium suksdorfii, Coriandrum sativum, Chrysanthemum morifolium and Swertia franchetiana [47,. Capryl aldehyde and methyl-n-nonyl ketone obtained from Houttuyniu cordata directly inhibit the RT enzyme [66]. Calanolides A (3) and B (4) [89] have been obtained from the plant Calophyllum inophyllum. The introduction of bulky groups has been shown to be essential at the C-4 position to enhance anti-HIV activity. The stereochemistry of the C-12 hydroxyl (R or S configured) is not, however, as critical for activity. Methyl groups at the C-10 and C-11positions were also shown to be required for activity. Hydrogen bond acceptors at C-12 were also shown to be responsible for the activity, both in calanolides and inophyllums. In vitro assay results revealed that (+)-Calanolide-A inhibits RT in two diverse template primer systems. The action of (+)-Calanolide-A is possible due to the bi-bi prearranged mechanism of RT. Calanolide is at least partially competitive about dNTP binding. Structure-activity-relationships and important key findings of Calanolides are shown in Figure 4. OH SAR features 1) Introduction of bulky substituents are essential at the C-4 position enhance anti-HIV activity.
2) Methyl groups at the C-10 and C-11 positios are also required for activity.
3) A hydrogen bond acceptor at the C-12 position is also resposible for activity.
4) The stereochemistry of the C-12 hydroxyl is not as critical. It can be R or S cofigured.

Critical for anti-HIV activity
Key Findings for mechanistic insights 1) Mechanistic in vitro assays reveal that Compound 3 inhibits RT in two different template primer systems.
2) The action of Compound 3 is likely due to the bi-bi ordered mechanism of RT.
3) Calanolide is at least partly competitive with respect to dNTP binding.
Catechin (21), suramin and protocatechuic acid (22) were shown to be the components identified from the plant extract and were considered to act on the integrase enzyme of HIV, thus prohibiting viral replication [139]. Compound 21 interacted with Thr66, Gly148, and Glu152 in the core domain of the enzyme, whereas compound 22 interacted with Thr66, His67, Glu152, Asn155, and Lys159. Some ribosome-inactivating proteins are considered to act on the integrase enzyme [141]. It was observed that compound 20, having a galloyl moiety, possessed the most potent activity due to its strong binding with amino acids of the integrase enzyme. In compound 19, the catechol group was partly responsible for the activity. Since compound 19 contains sugar moiety as well, which increases the solubility of the molecule, this enhances its activity. A ribosome-inactivating protein (RIP) named MAP30, which has been extracted from Momordica charantia, has been reported to act against HIV and cancer [142,143]. Zhao et al. discovered another RIP trichosanthin, from the roots of Trichosanthes kirilowii, which showed inhibitory activity against the integrase enzyme [144]. A variety of plant RIPs including agrostin, saporin, R-momorcharin, gelonin, α-momorchain, trichosanthin and luffin have also exhibited an inhibitory effect on HIV replication [145].

Structure-activity-relationships
1) Compound 20 has the galloyl moiety and possessed the most potent activity. This is due to its strong binding with amino acids of the integrase enzyme.
2) In compound 19, catechol part is responsible for the activity.
3) Compound 19 also contains a sugar moiety, which increases the solubilty of the molecule and thus enhancing its activity.
Molecules 2020, 25, x FOR PEER REVIEW 13 of 50 effective immunomodulatory action against HIV. From saponins, asiaticoside obtained from Centella asiatica [178] and glycyrrhizin from the roots of Glycyrrhiza glabra [179] have shown significant immunomodulatory activities.

Plants with Antioxidant Potential
In AIDS, many reactive oxygen species (ROS) have been produced due to the alteration in the levels of antioxidant enzymes [180]. This further leads to the damage of DNA and lipid peroxidation [181]. ROS can also stimulate the nuclear factor kappa B (NF-κB factor) which helps in the transcription of HIV and thus promote its replication [182]. Antioxidants are agents that reduce the levels of ROS and protect cellular DNA. N-Acetylcysteine is reported to acts as an antioxidant and inturn used in themanagement of HIV infection [183]. Various other antioxidants like selenium, lipoic acid, vitamin C, β-carotene and vitamin E have been utilized for the same purpose [184,185]. The antioxidants Cyanidin-3-glucoside (35) and peonidin (36), which are obtained from blackberries, have also been shown toslow down AIDS infection ( Figure 9) [159,186].

Plants with Antioxidant Potential
In AIDS, many reactive oxygen species (ROS) have been produced due to the alteration in the levels of antioxidant enzymes [180]. This further leads to the damage of DNA and lipid peroxidation [181]. ROS can also stimulate the nuclear factor kappa B (NF-κB factor) which helps in the transcription of HIV and thus promote its replication [182]. Antioxidants are agents that reduce the levels of ROS and protect cellular DNA. N-Acetylcysteine is reported to acts as an antioxidant and inturn used in themanagement of HIV infection [183]. Various other antioxidants like selenium, lipoic acid, vitamin C, β-carotene and vitamin E have been utilized for the same purpose [184,185]. The antioxidants Cyanidin-3-glucoside (35) and peonidin (36), which are obtained from blackberries, have also been shown to slow down AIDS infection ( Figure 9) [159,186].

Alkaloids
Alkaloids are the basic nitrogen-containing secondary metabolites in plants, active against many pathogens, including HIV. Buchapine is a quinolone alkaloid obtained from Eodia roxburghiana, which has shown activity against HIV [194]. From the roots of Tripterygium hypoglaucum, various alkaloidal compounds have been isolated, e.g., hypoglaumine B, triptonine A (37) and B [159], which exhibited anti-HIV potential and have potential for antiretroviral therapy [195]. Nitidine is another alkaloid that was isolated from plant roots of Toddalia asiatica, and has shown efficacy against HIV [196]. From the plant Symplocos setchuensis, the alkaloid harman (38) and another compound matairesinoside (39) were isolated andshowed potential for antiretroviral therapy due to their anti-HIV potential. Compound 39 acts on the viral replication enzymes, thus inhibiting HIV replication [197]. Another aromatic alkaloid polycitone A, from marine source Polycitor sp., exhibited potential activity against the reverse transcriptase of HIV. Hence, it efficiently inhibits HIV replication. Several other marine sponges have acted against the virus as well as other bacterial diseases [198]. The alkaloid 1-methoxy canthionone was reported from Leitneria floridana, and exhibited anti-HIV property [199]. Papaverine was obtained from Papaver sominiferum, and inhibited HIV replication [39]. Norisoboldine and corydine are two alkaloids obtained from the leaves of Croton echinocarpus, showing anti-HIV activity [200]. Table 2

Terpenoids
Terpenoids are the secondary metabolites that are derived from the isoprene unit (C 5 H 8 ). Terpenoids are the most abundant plant-based secondary metabolites and several compounds from this class have been derived from plants and found useful for their therapeutic potential [210,219,220]. Examples of terpenoids that have exhibited inhibition of HIV replication include betulinic acid, oleanolic acid, and platanic acid from Syzigium claviflorum leaves [221]. Celasdin B (51), is a triterpene from Celastrus hindsii (Celastraceae), reported inhibiting the HIV replication [222]. Prostratin from Homalanthus nutans (Euphorbiaceae) has also expressed significant anti-HIV activities [223]. From the stem bark of Garcinia speciosa, some anti-HIV therapeutic constituents have been isolated viz; garcisaterpenes A and C and theprotostanes. These compounds have been found to inhibit the activity of HIV reverse transcriptase and thus stop the HIV life cycle [224]. Maslinic acid (52), a terpenoid compound obtained from Geum japonicum also acts against the HIV protease enzyme [225]. From the stems and roots of plant Kadsura lancilimba, another triterpene lancilactone C (53) has been isolated whch is used to restrict the viral replication [226]. Oleanolic acid is the main terpenoid isolated from many plant species including Xanthoceras sorbifolia (Sapindaceae). The compound is known to inhibit HIV replication and play an important role in the treatment of AIDS [227]. Suberosol (54) is a lanostane type triterpenoid from the leaves of Polyalthia suberosa (Annonaceae) known toact through the same mechanism [228]. Another phorbol diester from Croton tiglium (Euphorbiaceae), 12-O-tetradecanoylphorbol-13-acetate, exhibited anti-HIV activity [223]. A Brazilian alga isolated from Dictyota pfaffii, from which an active diterpene component 8,10,18-trihydroxy-2,6-dolabelladiene has been isolated and has shown inhibitory activity of HIV reverse transcriptase [229,230]. A butenolide triterpene known as 3-epi-litsenolide has been articulated significant anti-HIV activity and was extracted from Litsea verticilla [231]. The alga Dictyota menstrualis is an important source for various diterpenes that exhibit HIV reverse transcriptase inhibition potential [232]. From the roots and rhizomes of plant Clausena excavate, a limonoid terpene named clausenolide-1-ethyl ether has shown potential for antiretroviral therapy [233]. Glycyrrhizin from the Glycyrrhiza glabra roots is another saponin terpenoid that showed anti-HIV activity by inhibiting the viral life cycle [234]. Oleanolic acid is a potent anti-HIV compound and is widely distributed in various plants including the leaves of Rosa woodsii, the leaves of Syzygium claviflorum, the aerial parts of Ternstromia gymnanthera, and the whole plants Hyptis capitata and Phoradendron juniperinum [227]. 12-Deoxyphorbol-13-phenylacetate, a phorbol ester from Euphorbia poissonii, has been reported for possessing anti-tumour activity and recently, it has potential for antiretroviral therapy because of its anti-HIV activity [235].

Key Findings
The heterocyclic ring is mainly resposible for the therapeutic activity. . Figure 11. More alkaloidal compounds possessing anti-HIV activity.

Flavonoids
Flavonoids are well-known phytoconstituents reported to exhibit several antiviral and antioxidant properties [278]. Flavonoids like quercetin 3-O-(2-galloyl)-L-arbinopyranose and gallate ester from Acer okamotoanum (Aceraceae), exhibited significant activity against integrase of HIV [279]. Xanthohumol (66), an important flavonoid from Humulus lupulus, has shown anti-HIV activity [280]. The flavonoid moiety (4H-chromen-4-one) is known to be mainly responsible for the therapeutic activity, while glycosidic portion attached to the flavonoid enhances the solubility of the compounds and thus boosts its therapeutic activity. Two flavonoids 6,8-diprenylkaempferol and 6,8-diprenylaromadendrin isolated from Monotes africanus have expressed potential activity against the AIDS virus [281]. Another anti-HIV biflavonoid named wikstrol B (67) (Figure 14) has been isolated from Wikstroemia indica (Thymelaeaceae) roots [282]. Baicalin is a flavonoid compound that inhibits HIV replication and is derived from Scutellaria baicalensis [282]. From the twigs and leaves of the medicinal plant Rhus succedanea, various anti-HIV flavonoids (robustaflavone, biflavonoids, and hinokiflavone) have been reported to act on the polymerase of the reverse transcriptase of HIV-1 [283,284]. 2-methoxy-3-methyl-4,6-dihydroxy-5-(3′-hydroxy)-cinnamoylbenzaldehyde, a chalcone flavonoid that has been extracted from Desmos sp. roots and exhibited strong activity against HIV-1 Several naturally obtained flavonoids, e.g., chrysin, epigallocatechin gallate (68) and quercetin (69) have been reported to show potent inhibitory activities against the replication of HIV [276,277]. The flavonoids Thalassiolin A, B and C from the grass Thalassia testudinum acted against HIV integrase, which inturn inhibited the life cycle of HIV-1. Thalassiolin A was found to be the most potent compound which inhibits the terminal cleavage [287][288][289]. Some biflavonoids, e.g., 2 ,3 -dihydroochnaflavone 7 -O-methylether and ochnaflavone 7 -O-methyl ether from Ochna integerrima, have shown moderate to weak anti-HIV activities [290,291]. Taxifolin (70), also known as dihydroquercetin, is mostly found the stems of Juglans mandshurica, and expressed strong inhibitory activity on the reverse transcriptase enzyme of HIV and thus plays a role in the prevention of HIV replication [292]. From Chrysanthemum morifolium flowers, two important flavonoids apigenin-7-O-β-D-(4 -caffeoyl)glucuronide and glucuronide have been isolated, which exhibited significant activity against the integrase of HIV-1 [293]. Mentha longifolia is another plant whose methanolic extracts are used for the isolation of several therapeutic flavonoids those were found to be active through the same mechanism [294].  [285]. Another chalcone, Hydroxypanduratin A, from the rhizomes of Boesenbergia pandurata depicted its action on the HIV protease enzyme [286].  Several naturally obtained flavonoids, e.g., chrysin, epigallocatechin gallate (68) and quercetin (69) have been reported to show potent inhibitory activitiesagainst the replication of HIV [276,277]. The flavonoids Thalassiolin A, B and C from the grass Thalassia testudinum acted against HIV integrase, which inturn inhibited the life cycle of HIV-1. Thalassiolin A was found to be the most potent compound which inhibits the terminal cleavage [287][288][289]. Some biflavonoids, e.g., 2″,3″-dihydroochnaflavone 7″-O-methylether and ochnaflavone 7′′-O-methyl ether from Ochna integerrima, have shown moderate to weak anti-HIV activities [290,291]. Taxifolin (70), also known as dihydroquercetin, is mostly found the stems of Juglans mandshurica, and expressed strong inhibitory activity on the reverse transcriptase enzyme of HIV and thus plays a role in the prevention of HIV replication [292]. From Chrysanthemum morifolium flowers, two important flavonoids apigenin-7-O-β -D-(4′-caffeoyl)glucuronide and glucuronide have been isolated, which exhibited significant activity against the integrase of HIV-1 [293]. Mentha longifolia is another plant whose methanolic extracts are used for the isolation of several therapeutic flavonoids those were found to be active through the same mechanism [294]. Compound 70 was demonstrated to inhibitthe activity of HIV-1 replication. Several other flavonoids such as flemiphyllin, formosanatin C (71), euchretin I (72) and quercetin are

Coumarins
Calanolides are a group of coumarins that act as non-nucleoside reverse transcriptase inhibitors and are derived from plants of the genus Calophyllum (Clusiaceae) [300]. The coumarin (+)-Calanolide A has already been subjected to in vivo studies and up to phase II clinical trials in healthy, HIV-negative subjects. These studies revealthat (+)-calanolide A has a favourable safety profile in humans as well as in animals [301,302], while calanolide B alongwith its derivative known as 7,8-dihydrocalanolide B from the plant Calophyllum lanigerum, showedsignificant anti-HIV potential based on cytopathogenic results of HIV on the cells of the host [300]. Another coumarin named suksdorfin (74) [303,304] isolated from the fruits of Lomatium suksdorfii belonging to the family Apiaceae, which has expressed inhibitory property on the HIV replication [303]. The compounds Cordatolides A and B from Calophyllum cordato-oblongum, were similar in structureto the Calanolides and were found to inhibit the replication of the HIV [300]. The coumarin skeleton is essential for anti-HIV activity ( Figure 15). Other coumarins like heraclenol (75) and heraclenin (76) exhibit IC 50 value of 20.1 µg/mL against H9 lymphocytes, while imperatorin (77) from the roots of Ferula sumbul falls under the same therapeutic category [305]. Bulky groups at the C-4 are also required to retain the anti-HIV activity, which is present in the prototype of a molecule like (+)-Calanolide-A. (+)-Calanolide-A is the most potent compound when compared with Cordatolide A (less active and devoid of the bulky group at the C-4 position). Several furanocoumarins (e.g., bergapten (78) and psolaren) from the roots of Prangos tschimganica, have exhibited significant activities against the HIV virus [306]. Mesuol (79) is another coumarin (from the category 4-phenylcoumarin) reported to inhibit the replication of HIV-1through the prohibition of the reverse transcription and phosphorylation of HIV [307]. A semisynthetic derivative of calanolide (known as oxocalanolide) was alsoreported to act efficiently against HIV [308]. Various furanocoumarins (e.g., imperatorin, xanthotoxin and xanthotoxol) have been extracted from the Aegle marmelos fruits [121,122]. The stem, roots, fruits, leaves, seeds and bark of the A. marmelous showed variable antiviral effects and have played an important role in Ayurvedic medicine. Imperatorin (77) is reported to exhibit about 60% inhibition of HIV-RT. The absence of a prenyl group resulted in the observed weak activity. This is exemplified in the cases of other furanocoumarins xanthotoxin (80) and xanthotoxol (81), shown in Figure 15 [309,310]. Coumarin Coumarin Figure 15. Coumarins with significant Anti-HIV potential.

Proteins
Proteins are the amino acid-containing plant components that usually contain Figure 15. Coumarins with significant Anti-HIV potential.

Proteins
Proteins are the amino acid-containing plant components that usually contain ribosome-inactivating proteins as well as lectins [311]. A plant protein called MAP30 from Momordica charantia is known to possess anticancer potential along with anti-HIV properties [312]. Various plant ribosome-inactivating proteins have been identified for their anti-HIV activities. Trichosanthin is a ribosome-inactivating protein isolated from Trichosanthes kirilowii that has shown anti-HIV activity [313]. Various plant ribosome-inactivating proteins have been identified for their anti-HIV activities, e.g., an anti-HIV ribosome-inactivating protein balsamin has been extracted from Momordica balsamina [314]. Pf-gp6 is another protein reported from Perilla frutescens which has exhibited an inhibitory action on HIV replication [315]. Some ribosome-inactivating proteins known as Pokeweed antiviral proteins have been separated from a pokeweed plant (Phytolacca americana) and have expressed efficient anti-HIV activities [316]. A list of plant proteins has been given in Table 4, along with their botanical sources. Tannins are mainly categorized into gallotannins and ellagitannins. While gallotannins are hydrolysable and contain gallic acid polyesters ellagitannins are non-hydrolyzable, so-called condensed tannins conatining hexahydroxydiphenic acids, i.e., flavan-3-ol (proanthocyanidins) moieties [345,346]. Corilagin (82) and Geraniin (83) (Figure 16), from roots of Phyllanthus amarus, are two ellagitannins that possess anti-HIV activities [347]. Besides, a proanthocyanidin compound from the plant Cupressus sempervirens, exerted anti-HIV properties [348]. Catechins, the polyphenols that are obtained from green tea, and theaflavins (e.g., compound 84) isolated from black tea, possess antiviral activity. Theaflavins and their derivatives are potent inhibitors of HIV replication [349]. Compounds 82 and 83 blocked the interaction of HIV-1 gp120 with its primary cellular receptor CD4. Besides, the observed results showed that compound 83 exhibited inhibitory effects on HIV, not only in vitro but also in vivo. Compound 84 inhibited HIV-1 entry into target cells by blocking the HIV-1 envelope glycoprotein-mediated membrane fusion. The ability of this compound to block the formation of the gp41 six-helix bundle was determined using Fluorescence native polyacrylamide gel electrophoresis, while detection of the binding of gp120 to CD4 was done by ELISA. Molecular docking analyses suggested that compound 84 may bind with to the highly conserved hydrophobic pocket on the surface of the central trimeric coiled-coil of gp41.
Molecules 2020, 25, x FOR PEER REVIEW 26 of 50 moieties [345,346]. Corilagin (82) and Geraniin (83) (Figure 16), from roots of Phyllanthus amarus, are two ellagitannins that possess anti-HIV activities [347]. Besides, a proanthocyanidin compound from the plant Cupressus sempervirens, exerted anti-HIV properties [348]. Catechins, the polyphenols that are obtained from green tea, and theaflavins (e.g., compound84) isolated from black tea, possessantiviral activity. Theaflavins and their derivatives are potent inhibitors of HIV replication [349]. Compounds 82 and 83 blocked the interaction of HIV-1 gp120 with its primary cellular receptor CD4. Besides, the observed results showed that compound 83 exhibited inhibitory effects on HIV, not only in vitro but also in vivo.Compound 84 inhibited HIV-1 entry into target cells by blocking the HIV-1 envelope glycoprotein-mediated membrane fusion. The ability of this compound to block the formation of the gp41 six-helix bundle was determined using Fluorescence native polyacrylamide gel electrophoresis, while detection of the binding of gp120 to CD4 was done by ELISA. Molecular docking analyses suggested that compound 84 may bind with to the highly conserved hydrophobic pocket on the surface of the central trimeric coiled-coil of gp41.
[IC 50  2) Having the ability to block the formation of the gp41 six-helix bundle was determined using fluorescence native polyacrylamide gel electrophoresis.
3) Detection of the binding of gp120 to CD4 by ELISA.

Lignans
Several extensive reports on plant-based lignans which have shown strong activities against viral diseases, including AIDS, exist [350]. Several lignans like anolignan A (85) and anolignan B, alongwith dibenzylbutadiene lignans have been isolated from Anogeissus acuminate and have exhibited significant activity against HIV [351]. From Phyllanthus myrtifolius(Euphorbiaceae), phyllamyricin D (86) and phyllamyricin F (87) (Figure 17) were isolated and shown to possess inhibitory activity against the HIV-RT enzyme [352]. The benzoaryl moiety was proven to be essential for the anti-HIV activity of lignans. This group is responsible for inhibiting HIV replication. Gomisin is another example of lignan isolated from Kadsura interior and showed potent inhibitory activity against the RT enzyme of HIV [353]. From Arnebia euchroma, some caffeic acid isomers have been evaluated but have only expressed weak activities against HIV replication [354]. The compound 2-hydroxy-2 (3′,4′-dihydroxyphenyl)-methyl-3-(3″,4″-dimethoxyphenyl) methyl γ-butyrolactone is a dibenzylbutyrolactone type lignan from Phenax angustifolius with established anti-HIV activity [355]. From Schisandra rubriflora fruits, other dibenzocyclooctadiene type lignans (rubrisandrin A and rubrisandrin B) have been isolated having anti-HIV activities [356].

Responsible for activity
Key Findings 1) Anti-HIV activity using anti-HIV assay towards HOG.R5 cells.

Conclusions
Plants are known to exhibit a huge repertoire of bioactive metabolites [436]. A significant number of reports on the capability of natural compounds with potential as anti-HIV agents have appeared during the last few decades. This review article presents the rational approaches for the design of therapeutic potential candidates as anti-HIV agents. Even though there have been many extensive achievements in the field of HIV chemotherapy, there remains a great demand for novel lead compounds for anti-HIV drug discovery and drug development. Numerous plant species have been evaluated for their inhibitory activities on the essential HIV enzymes such as RT, protease, and integrase, which play an important role in HIV replication. Several secondary metabolites have been extracted from the various parts of plantsthat act as potent anti-HIV agents via different mechanisms of action.
Therapeutically active compounds from plants can also aid as necessary leads for the discovery and development of novel and more potent compounds that can be derived synthetically. For instance, synthetic ingenol compounds have been derived based on naturally occurring compound Ingenol and a variety of synthetic derivatives have been evolved from the naturally occurring compound Artemisinin, which exhibits significant anti-HIV activitiesof potential scaffolds from them for the complete eradication of HIV/AIDS. A recent review has attempted to show themost successful medical therapeutics derived from natural products, including those studied in the field of HIV/AIDS [437]. Besides, computer-aided (virtual) [438] and large-scale in vitro screening [439] approaches have recently been carried out on natural compound libraries to identify natural products with anti-HIV properties. Novel therapeutic approaches have been attempted, including searching for new HIV-1 latency-reversing agents, i.e., compounds not only capable of HIV suppression but also eliminating HIV reservoirs [440,441].