Phytochemicals Derived from Catharanthus Roseus and Their Health Beneﬁts

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Review Phytochemicals Derived from Catharanthus roseus and Their Health Beneﬁts

Hong Ngoc Thuy Pham 1,2,* , Quan Van Vuong 1, Michael C. Bowyer 1 and Christopher J. Scarlett 1,*

1 School of Environmental and Life Sciences, Faculty of Science, University of Newcastle, Ourimbah, NSW 2258, Australia; [email protected] (Q.V.V.); [email protected] (M.C.B.) 2 Faculty of Food Technology, Nha Trang University, No. 2 Nguyen Dinh Chieu Street, Nha Trang City 650000, Vietnam * Correspondence: [email protected] (H.N.T.P.); [email protected] (C.J.S.)

Received: 18 November 2020; Accepted: 17 December 2020; Published: 21 December 2020

Abstract: Catharanthus roseus (C. roseus) is an important medicinal plant distributed in many countries. It has attracted increasing attention due to it being shown to possess a range of phytochemicals with various biological activities such as antioxidant, antibacterial, antifungal, antidiabetic and anticancer properties. Remarkably, vinblastine and vincristine isolated from this plant were the first plant-derived anticancer agents deployed for clinical use. Recently, new isolated indole alkaloids from this plant including catharoseumine, 140,150-didehydrocyclovinblastine, 17-deacetoxycyclovinblastine and 17-deacetoxyvinamidine effectively inhibited human cancer cell lines in vitro. Moreover, vindoline, vindolidine, vindolicine and vindolinine isolated from C. roseus leaf exhibited in vitro antidiabetic property. These findings strongly indicate that this plant is still a promising source of bioactive compounds, which should be further investigated. This paper provides an overview of the traditional use and phytochemical profiles of C. roseus, and summarises updated techniques of the preparation of dried material, extraction and isolation of bioactive compounds from this plant. In addition, purported health benefits of the extracts and bioactive compounds derived from this plant were also addressed to support their potential as therapeutic agents.

Keywords: Catharanthus roseus; bioactive compounds; phytochemicals; health beneﬁts

1. Introduction Natural resources including herbal plants, which contain a large variety of phytochemicals promising as traditional medicine to treat chronic and infectious diseases, have been considered as safe and effective alternatives with fewer side effects compared to synthetic agents [1]. Amongst the plethora of medicinal plants identified, Catharanthus roseus (L.) G. Don (C. roseus) has been widely used to treat various diseases in many countries. The hot water extract of the dried C. roseus leaf has been used for the treatment of diabetes in Jamaica, Kenya and the West Indies or the hot water extract of the dried plant has been taken orally as complementary and alternative therapies for various types of cancers, heart disease and leishmaniasis in Peru [2]. More scientific evidence has proved the potential health benefits of individual phytochemicals extracted from this plant. Of note, vinblastine and vincristine from C. roseus, and their synthetic analogues, have been used in combination with other cancer chemotherapeutic drugs for treating advanced testicular cancer, breast and lung cancers [3]. The drying and extraction process are crucial steps prior to isolation and purification of bioactive compounds from plant material. Drying aims to remove moisture content and reduce water activity in plant material, thus inactivating the enzymes responsible for degrading many bioactive compounds,

Technologies 2020, 8, 80; doi:10.3390/technologies8040080 www.mdpi.com/journal/technologies Technologies 2020, 8, x FOR PEER REVIEW 2 of 16 Technologies 2020, 8, 80 2 of 16 in plant material, thus inactivating the enzymes responsible for degrading many bioactive compounds, decreasing the rate of microbial growth and reducing the costs of transportation and decreasing the rate of microbial growth and reducing the costs of transportation and preservation. preservation. However, the drying treatment has been found to have a significant effect on the However, the drying treatment has been found to have a significant effect on the retention of bioactive retention of bioactive compounds and antioxidant capacity of plant material [4–7]. Therefore, compounds and antioxidant capacity of plant material [4–7]. Therefore, selecting a suitable drying selecting a suitable drying method is very important to maintain the high yield of bioactive method is very important to maintain the high yield of bioactive compounds and antioxidant power compounds and antioxidant power within plant material; however, the economic aspect and the within plant material; however, the economic aspect and the accessibility of drying equipment should accessibility of drying equipment should also be considered. Similarly, the extraction process is also also be considered. Similarly, the extraction process is also a key step to obtain bioactive compounds a key step to obtain bioactive compounds from plant materials. Extraction techniques have been from plant materials. Extraction techniques have been reported to significantly affect the extraction reported to significantly affect the extraction efficiency and stability of bioactive compounds [8–11]. efficiency and stability of bioactive compounds [8–11]. Hence, this paper provides a comprehensive Hence, this paper provides a comprehensive review of different techniques of drying, extracting and review of different techniques of drying, extracting and isolating bioactive compounds from the target isolating bioactive compounds from the target plant material, C. roseus, and discusses its potential plant material, C. roseus, and discusses its potential health benefits along with its traditional use. health benefits along with its traditional use. 2. Taxonomy and Traditional Use of C. roseus 2. Taxonomy and Traditional Use of C. roseus C. roseus is native to Madagascar and known as the Madagascar periwinkle. It is now grown in many countriesC. roseus and is anative common to Madagascar decorative, easyand known growing as and the spreadingMadagascar perennial periwinkle. herb. It The is nowC. roseus grownplant in manyis 30–60 countries cm tall withand youngis a common pubescent decorative, branches. ea Itssy leavesgrowing are and oblong spreading or oblanceolate, perennial membranous,herb. The C. roseusentire, plant obtuse is or30–60 mucronate, cm tall with and haveyoung short pubescent petioles branches. (Figure1 ).Its Inflorescence leaves are oblong occurs or at oblanceolate, the axillary withmembranous, 1–4 flowered entire, cymes, obtuse and or flowers mucronate, are from and whitehave short to red petioles depending (Figure on the 1). cultivar.Inflorescence The calyx occurs tube at theis short, axillary but with the corolla 1–4 flowered tube is long,cymes, sparsely and flowers pubescent are from above white and theto red throat depending is hairy withinon the orcultivar. below Thethe stamens.calyx tube The is short, ovary but is pubescent, the corolla long tube and is long, the stigma sparsely is pentagonal.pubescent above Its fruits and are the 15–25 throat mm is hairy long withinand have or below two follicles the stamens. [12]. The ovary is pubescent, long and the stigma is pentagonal. Its fruits are 15–25 mm long and have two follicles [12].

Figure 1. CatharanthusCatharanthus roseus (L.)(L.) G. Don (Patricia White cultivar) (Pham, 2014).

This plant has been used as a traditional medicine in many countries (Table 1 1).). TheThe drieddried leafleaf oror entire plantplant isis boiled boiled with with water water and and then then the the extract extrac ist taken is taken orally orally to treat to treat diabetes diabetes in Northeast in Northeast India, India,the Cook the Islands, Cook Islands, Australia, Australia, England, England, Thailand, Thailand, Natal, Mozambique, Natal, Mozambique, Philippines, Philippines, Dominican Dominican Republic, Jamaica,Republic, Northern Jamaica, EuropeNorthern and Europe Vietnam and [13 Vietnam–17]. The [13–17]. aqueous The extract aqueous of the extract leaf or of the the whole leaf or plant the wholeis also usedplant byis Cookalso Islandused by and Cook Vietnamese Island peopleand Vietnamese [13,17], and people Kenyans [13,17], as complementary and Kenyans and as complementaryalternative therapies and foralternative various typestherapies of cancer for various including types throat, of cancer stomach including and oesophageal throat, stomach cancers [and18]. Theoesophageal people in cancers the Kancheepuram [18]. The people District in the of Kanc Tamilheepuram Nadu, India District mix theof Tamil powder Nadu, of C. India roseus mixwhole the plantpowder with of C. cow’s roseus milk, whole which plant is takenwith cow's orally milk, to treat which diabetes is taken [19 ].orallyC. roseus to treatroot diabetes is dried, [19]. ground C. roseus and rootdecocted is dried, for curing ground urogenital and decocted infections for curing in the urogenital Venda region, infections South Africa in the [Venda20], gonorrhoea region, South in Limpopo Africa [20],Province, gonorrhoea South Africa in Limpopo [21] and Province, stomach South ache inAfrica the Mutirikwi [21] and stomach area of Zimbabwe ache in the [ 22Mutirikwi]. area of Zimbabwe [22].

Technologies 2020, 8, 80 3 of 16

Table 1. Traditional use of C. roseus in some countries.

Part Used Disease Preparation Mode of Administration Country References The whole plant is Kancheepuram Diabetes powdered and mixed with Oral intake district of Tamil [19] Whole plant, leaf cow’s milk. Nadu, India Diabetes The leaf is boiled with water. Oral intake Northeast India [14] Diabetes Northern Leaf The dried leaf is decocted. Oral intake [16] mellitus Europe Leaf of purple or Diabetes, Eighteen leaves are boiled in white ﬂowered hypertension a kettle of water. The cool Oral intake Cook Island [13] varieties and cancer solution is drunk daily. Throat, Oral intake. Usually taken The whole plant is boiled stomach, together with Sesbania sesban Whole plant with water. Kenya [18] oesophageal whole plant. Pound cancer Applied topically Urogenital The root is air dried, ground Venda region, Root Oral intake [20] infections and decocted. South Africa Limpopo Root Gonorrhoea The root is boiled for 20 min. Oral intake Province, South [21] Africa Crushed roots are mixed Mutirikwi area Root Stomach Oral intake [22] with a cup of water. of Zimbabwe Diabetes, hypertension, The whole plant is boiled Whole plant Oral intake Vietnam [17] dysentery, with water. cancer

3. Major Bioactive Compounds Derived from C. roseus A range of alkaloids (nitrogen-containing organic compounds other than amino acids, peptides, purines and derivatives, amino sugars and antibiotics) [23] have been found in C. roseus (Table2). Alkaloids are broadly classiﬁed as heterocyclic or non-heterocyclic, depending on the chemical

Technologiesstructure. 2020 Heterocyclic, 8, x FOR PEERalkaloids REVIEW are those that contain the nitrogen atom in the ring system,4 of 17 the size of which leads to further subclassiﬁcation as pyrrole, pyrrolizidine, pyridine, piperidine, quinoline, isoquinoline,Technologies 2020, 8 norlupinane, x FOR PEER REVIEW orTable indole 2. Identified alkaloids. alkaloids Non-heterocyclic in C. roseus. alkaloids, which are also4 of 17 sometimes called proto-alkaloidsPlant or biological amines, are less commonly found in nature. These molecules have a TechnologiesAlkaloids 2020, 8, x FOR PEER REVIEW Quantity Chemical Structure Ref.4 of 17 nitrogen atom, whichPart is not a partTable of2. anyIdentified ring alkaloids system suchin C. roseus as ephedrine,. cathinone and colchicine [23]. Leaf 0.189−0.523 mg/g DE Plant Table 2. Identified alkaloids in C. roseus. OH VincristineAlkaloids Stem 0.082−0.388Quantity mg/g DE Chemical Structure Ref. Part Table 2. Identiﬁed alkaloids in C.N roseus. RootPlant 0.078−−0.659 mg/g DE Alkaloids Leaf 0.189 Quantity0.523 mg/g DE Chemical Structure Ref. − OH N AlkaloidsVincristine PlantLeafStemPart Part 0.2660.082 Quantity−1.2930.388 mg/g mg/g DE DE ChemicalH Structure References N N −− H StemLeaf 0.2850.189−1.0560.523 mg/g mg/g DE DE H O [24] Root 0.078 0.659 mg/g DE OH Leaf 0.189 0.523− mg/g DE MeO2C Vincristine Stem 0.082−0.388 mg/g DE H N OH Leaf 0.266− 1.293 mg/g DE N H O N Vinblastine − N MeO CO2Me Vincristine StemRoot 0.0820.0780.388−0.659 mg mg/g/g DE DE H Stem 0.285− 1.056 mg/g DE R H O [24] Root 0.463− −1.638 mg/g DE N Leaf 0.266 1.293 mg/g DE Vinchristine:MeO2C R =H CHO Vinblastine: R = CH H OH Root 0.078 0.659 mg/g DE N 3 O − H N [24] Vinblastine Stem 0.285− 1.056 mg/g DE MeO CO2MeO H [24] Leaf 0.266 1.293− mg/g DE MeO2C R Root 0.463 1.638 mg/g DE H OH Leaf 0.001−−0.006 mg/g DE Vinchristine: R = CHO O Vinblastine Vinblastine: R = CH N CO Me Vinblastine Stem 0.285 −1.056 mg/g DE MeO 3 2 Stem 0.001− −0.007 mg/g DE H NR Root 0.463 1.638 mg/g DE Vinchristine: R = CHO Root 0.463 1.638 mg/g DE Vinblastine: R = CH Leaf 0.001−0.006 mg/g DE 3 − N Vinpocetine − [24] Stem 0.001 0.007 mg/g DE N LeafRoot 0.0010.001−0.006−0.056 mg mg/g/g DE DE H Leaf 0.001− 0.006 mg/g DE O − NO VinpocetineVinpocetine Stem 0.001 0.007 mg/g DE H N [24] [24] Stem 0.001 0.007 mg/g DE Root 0.001− −0.056 mg/g DE − O Vinpocetine RootLeaf 0.0010.001 0.0560.036 mg mg/g/g DE DE N [24] O Stem 0.003−−−0.055 mg/g DE Root 0.001 0.056 mg/g DE O OCH3 − O LeafLeaf 0.0010.0010.0360.036 mg mg/g/g DE DE H O − N Reserpine − OCH3 [24] Reserpine Stem 0.003 0.055 mg/g DE O H OCH [24] −− CH O N 3 StemRootLeaf 0.0030.0010.0010.0550.0360.036 mg mg/g mg/g/g DE DE DE 3 H OCH H O 3 − N H Stem 0.003−0.055 mg/g DE MeO C OCH3 Reserpine Root 0.001 0.036 mg/g DE 2 OCH3 [24] OCH3 − O − N H O Root 0.001 0.036 mg/g DE CH3O H N H − H OCH3 Reserpine Leaf 0.165 0.970 mg/g DE OCH3 [24] MeO C OCH3 2 O −− N H H StemRoot 0.1620.001 5.4870.036 mg/g mg/g DE DE CH3O H N H OCH3 MeO C OCH3 Leaf 0.165−0.970 mg/g DE 2 O N H Ajmalicine H [24] Stem 0.162−5.487 mg/g DE H H Root 0.124−17.675 mg/g DE N Leaf 0.165−0.970 mg/g DE H 3CO O O − N H H Ajmalicine Stem 0.162 5.487 mg/g DE H N [24] H Root 0.124−17.675 mg/g DE − H3CO O Leaf 0.016 0.067 mg/g DE N H Ajmalicine H O [24] H OH Stem 0.025−0.085 mg/g DE OH N Root 0.124 17.675 mg/g DE H 3CO Ajmaline Leaf 0.016−0.067 mg/g DE O [24] N H − OH Root 0.036 −0.140 mg/g DE OH N Stem 0.025 0.085 mg/g DE CH3 Leaf 0.016−0.067 mg/g DE Ajmaline OH [24] − N OHH Stem 0.025−−0.085 mg/g DE N LeafRoot 0.1390.036 0.5390.140 mg/g mg/g DE DE CH Ajmaline − 3 [24] Stem 0.185 1.572 mg/g DE N H N H Root 0.036−0.140 mg/g DE Leaf 0.139−0.539 mg/g DE CH3 Yohimbine N [24] − H RootStem 0.3160.185−2.4331.572 mg/g mg/g DE DE NH H Leaf 0.139−0.539 mg/g DE MeO2C OH − Yohimbine Stem 0.185 1.572 mg/g DE N N [24] H H − H LeafRoot 0.1390.316−2.9782.433 mg/g mg/g DE DE MeO C Yohimbine N 2 OH [24] Stem 1.754−2.302 mg/g DE H Root 0.316−2.433 mg/g DE H Leaf 0.139−2.978 mg/g DE MeO2C OH N Stem 1.754−2.302 mg/g DE OH − NH Leaf 0.139 2.978 mg/g DE CO Me Vindesine 2 OH [24] Stem 1.754−2.302 mg/g DE − N NH2 Root 1.552 3.247 mg/g DE OH NH N N CO Me O Vindesine 2 OH [24] CH O N CH3 3 OH Root 1.552−3.247 mg/g DE NH NH2 CO Me Vindesine 2 N OH [24] N OH O − CH3 NH Root 1.552 3.247 mg/g DE CH3O 2 Leaf 2.868−22.079 mg/g DE Serpentine N [24] − N O Stem 11.265 50.078 mg/g DE CH3 OHCH3O Leaf 2.868−22.079 mg/g DE Serpentine [24] Stem 11.265−50.078 mg/g DE OH Leaf 2.868−22.079 mg/g DE Serpentine [24] Stem 11.265−50.078 mg/g DE

Technologies 2020, 8, x FOR PEER REVIEW 4 of 17

Technologies 2020, 8, x FOR PEER REVIEWTable 2. Identified alkaloids in C. roseus. 4 of 17 Technologies 2020, 8, x FOR PEERPlant REVIEW 4 of 17 Alkaloids Table 2.Quantity Identified alkaloids in C. roseusChemical. Structure Ref. Technologies 2020, 8, x FOR PEERPart REVIEW 4 of 17 − PlantLeaf Table0.189 2.0.523 Identified mg/g alkaloidsDE in C. roseus. Alkaloids Quantity ChemicalOH Structure Ref. Vincristine StemPart Table0.082 2.−0.388 Identified mg/g alkaloidsDE in C. roseus. Plant N Alkaloids Root 0.078−Quantity0.659 mg/g DE Chemical Structure Ref. LeafPart 0.189 0.523 mg/g DE Plant − OH N VincristineAlkaloids StemLeaf 0.0820.266−Quantity0.3881.293 mg/g DE ChemicalH Structure Ref. LeafPart 0.189 0.523 mg/g DE N N Stem 0.285−1.056 mg/g DE H OH O Root 0.078−0.659 mg/g DE H [24] Vincristine StemLeaf 0.0820.189 0.3880.523 mg/g DE MeO C 2 N HN Leaf 0.266−1.293 mg/g DE HOH OH Root 0.078−0.659 mg/g DE O VinblastineVincristine Stem 0.082 0.388 mg/g DE N N CO Me − MeON 2 Stem 0.285−1.056 mg/g DE H N O RootLeaf 0.2660.078−1.2930.659 mg/g DE H R H [24] Root 0.463 1.638 mg/g DE MeO C NVinchristine:2 R = CHOH − N OH Stem 0.285−1.056 mg/g DE HVinblastine: R = CH 3 O O Leaf 0.266 1.293 mg/g DE H N H [24] Vinblastine MeO CO2Me NMeO C 2 H Stem 0.285−1.056 mg/g DE H R OHO Root 0.463−1.638 mg/g DE H O [24] Vinblastine − Vinchristine: R = CHO N CO Me Leaf 0.001 0.006 mg/g DE MeO2MeOC 2 Vinblastine: R = CH 3 H OH − R O Vinblastine Root 0.463−1.638 mg/g DE N CO Me Stem 0.001 0.007 mg/g DE Vinchristine:MeO R = CHO H N 2 Vinblastine: R = CH Root 0.463−1.638 mg/g DE 3 R Leaf 0.001 0.006 mg/g DE Vinchristine: R = CHO Vinpocetine − Vinblastine: R =N CH 3 [24] Stem 0.001−0.007 mg/g DE H N RootLeaf 0.001−0.0060.056 mg/g DE − O StemLeaf 0.001−0.0070.006 mg/g DE H N Vinpocetine N O [24] − Stem 0.001−0.007 mg/g DE H N Root 0.001 0.056 mg/g DE N Vinpocetine Leaf 0.001−0.036 mg/g DE O [24] O Vinpocetine Root 0.001−0.056 mg/g DE N [24] Stem 0.003−0.055 mg/g DE O − O OCH3 Root 0.001 0.056 mg/g DE O Leaf 0.001−0.036 mg/g DE O N H O OCH Reserpine Stem 0.003−0.055 mg/g DE 3 [24] Leaf 0.001 0.036 mg/g DE O − N H OCH3 Root 0.001 0.036 mg/g DE CH3O H − H O OCH3 StemLeaf 0.0030.001−0.0550.036 mg/g DE N H OCH3 OCH Reserpine − MeO2C OCH3 3 [24] Stem 0.003 0.055 mg/g DE H O − N H N OCH Root 0.001 0.036 mg/g DE CH3O H OCH3 TechnologiesReserpine2020, 8, 80 H OCH3 3 [24] 4 of 16 O − H O Leaf 0.165 0.970 mg/g DE NMeO C OCH3 − N H 2 OCH Reserpine Root 0.001 0.036 mg/g DE CH3O H 3 [24] − H OCH3 Stem 0.162 5.487 mg/g DE N HO − N H MeO C OCH3 Root 0.001 0.036 mg/g DE CH3O H 2 − H OCH3 Leaf 0.165 0.970 mg/g DE O Table 2. Cont. NMeOH C OCH3 Ajmalicine − H 2 [24] Stem 0.162−5.487 mg/g DE N H H RootLeaf 0.1240.165−17.6750.970 mg/g mg/g DE DE Alkaloids Plant Part Quantity− ChemicalH Structure3CO References Stem 0.162−5.487 mg/g DE OH O Leaf 0.165 0.970 mg/g DE N H N Ajmalicine H [24] − H Stem 0.162− 5.487 mg/g DE N H O LeafRoot 0.1650.1240.97017.675 mg mg/g/g DE DE N HH3CO Ajmalicine − H [24] Leaf 0.016− 0.067 mg/g DE H O O Root 0.124−17.675 mg/g DE N OHH OH AjmalicineAjmalicine Stem 0.025−0.085 mg/g DE H H3CON [24] [24] Stem 0.162 5.487 mg/g DE H Root 0.124−−17.675 mg/g DE O Ajmaline Leaf 0.016−0.067 mg/g DE H 3CO [24] Root 0.124 17.675 mg/g DE N H O − OH OH StemRoot 0.0250.036− −0.0850.140 mg/g DE N Leaf 0.016 0.067 mg/g DE CH3 Leaf 0.016 0.067 mg/g DE OH Ajmaline Stem 0.025−0.085 mg/g DE OH N [24] Leaf 0.016− 0.067 mg/g DE N H − OH Ajmaline RootLeaf 0.0360.139−0.1400.539 mg/g DE OH Ajmaline StemStem 0.0250.0250.0850.085 mg mg/g/g DE DE CH3 N [24] [24] − − N H Stem 0.185−1.572 mg/g DE N H Ajmaline Root 0.036 0.140 mg/g DE CH [24] Root 0.036 0.140− mg/g DE N 3 H RootLeaf 0.1390.036− −0.5390.140 mg/g DE Yohimbine NCH3 [24] Stem 0.185−1.572 mg/g DE H LeafRootLeaf 0.1390.1390.3160.539−0.5392.433 mg mg/g/g DE DE N H H − − MeO2C OH StemLeaf 0.1850.139 1.5720.539 mg/g DE N H YohimbineYohimbine N [24] [24] Stem 0.185 1.572 mg/g DE H − H Technologies 2020, 8, x FOR PEERStemRoot REVIEW 0.3160.185 − 2.4331.572 mg/g DE N H 5 of 17 Yohimbine Leaf 0.139−2.978 mg/g DE N [24] Root 0.316 2.433 mg/g DE H MeO2C OH Technologies 2020, 8, x FOR PEER REVIEW − − H 5 of 17 Yohimbine RootStem 0.3161.754 2.4332.302 mg/g DE N [24] H MeO2C OH Technologies 2020, 8, x FOR PEERPlantRoot REVIEW 0.316 −2.433 mg/g DE H 5 of 17 Alkaloids Leaf 0.139 Quantity2.978 mg/g DE Chemical Structure Ref. Part MeO2C OH Technologies 2020, 8, x FOR PEERPlantStem REVIEW 1.754 −2.302 mg/g DE N 5 of 17 Alkaloids Leaf 0.139−Quantity2.978 mg/g DE ChemicalNH Structure OH Ref. PlantPart Technologies 2020, 8, x FOR LeafPEER REVIEW 0.139 2.978 mg/g DE CO Me 5 of 17 AlkaloidsVindesine StemLeaf 1.7540.139− −Quantity2.3022.978 mg/g DE Chemical2 Structure OH Ref.[24] Part N Vindesine Plant N NH Technologies 2020, 8, x FOR PEERRoot REVIEW 1.552 −3.247 mg/g DE 2 5 of 17 [24] Alkaloids Stem 1.754 Quantity2.302 mg/g DE ChemicalNH Structure OH Ref. PlantRootPart 4.927−49.851 mg/g DE N N O Alkaloids Quantity N ChemicalCON2Me StructureN O Ref. Vindesine H OH [24] N CH3 OH Part − NHCH3O O PlantRoot 4.927 −49.851 mg/g DE N N NH2 StemRoot 1.7541.5522.3023.247 mg mg/g/g DE DE CO Me MeO C VindesineAlkaloids Quantity Chemical2H Structure2 OHOH [24]Ref. RootPart 4.927−−49.851 mg/g DE NH N N O − N N O NH Root 1.552 3.247 mg/g DE OH CO2HMe MeO2C 2 Vindesine Root 1.552 3.247 mg/g DE N CH3 OH [24] CH3O Root 4.927−−49.851 mg/g DE N O Leaf 2.868 −22.079 mg/g DE N MeO C O NH2 Root 1.552 3.247 mg/g DE N H 2 Serpentine − N CH3 O [24] LeafRoot 2.8684.92722.079−49.851 mg mg/g/g DE DE CH3O N N Stem 11.265 50.078 mg/g DE H O − N OH MeON 2C − CH3 O Root 4.927−49.851 mg/g DE CH3O [25,26 Serpentine Leaf 2.868 22.079 mg/g DE N MeO2C [24] Catharanthine Stem Leaf 11.265 0.284350.078 ± 0.0132 mg/ gmg/g DE H N Serpentine − OH [24] − N [25,26] Stem 11.265− 50.078 mg/g DE MeON C Catharanthine RootLeaf 4.9272.868 0.284349.85122.079 ± 0.0132 mg mg/g/g mg/g DE DE OH CO2Me2 Serpentine CH [24] − −− N 3 [25,26] Catharanthine Stem Leaf 11.2652.868 0.284322.07950.078 ± 0.0132 mg/gmg/g mg/g DEDE CONMe 2 Serpentine NCH3 [24]] − N [25,26 Catharanthine Stem Leaf 11.265 0.2843 50.078± 0.0132 mg/g mg/g DE CO2Me Vindolidine Leaf 0.14% CH N 3 [25,26] [25,26] CatharanthineCatharanthine Leaf Leaf 0.2843 0.2843− ±0.0132 0.0132 mg mg/g/g N Vindolidine Leaf 5.301 19.463 0.14% mg/g DE CO2Me ± CHN ] N3 O [25,26 Catharanthine Leaf 5.301 0.2843−19.463 ± 0.0132 mg/g mg/g DE CO2Me Vindolidine Leaf 0.14% CH3 Stem 0.144−3.344 mg/g DE NN ] − H O O Vindolidine Leaf 5.301 19.463 0.14% mg/g DE CO2Me − CHN3 O [24,27 Stem 0.144 3.344 mg/g DE H O VindolidineVindoline Leaf 5.301−19.463 0.14% mg/g DE CO2Me ] Stem 0.144−3.344 mg/g DE R N H OH [24,27 − N H O O Vindolidine LeafLeaf 5.301 0.14%19.463 mg/g DE CO2Me VindolidineVindoline Leaf 0.14% CHN 3 [24,27] RootStem 0.0210.144−9.6903.344 mg/g DE R N H OH O H COOMe Vindoline Leaf 5.301−19.463 mg/g DE Vindolidine:CH R = H 2 ] Stem 0.144−3.344 mg/g DE R Vindoline:N 3 H R OH= OCH [24,27 Root 0.021−9.690 mg/g DE N H O 3 O Vindolidine: R = HCO Me [24,27] CH3 2 [24,27 Vindoline Leaf 5.301 19.463− mg/g DE Vindoline:N H ROH = OCH ] RootStem 0.0210.144 9.6903.344 mg/g DE R 3 − Vindolidine:H R = HCOO2Me Vindoline N ] R Vindoline:CH 3 H ROH = OCH [24,27 Vindoline StemRoot 0.1440.0213.344−9.690 mg mg/g/g DE DE 3 CH3O CH3O − Vindolidine:CH3 R = HCO2Me Vindoline − N H OH ] RootRoot 0.0210.0219.6909.690 mg mg/g/g DE DE R Vindoline: R = OCH3 CHVindolidine:3O CH R3O = H − CH CH − H3C Vindoline: 3 R = OCH3 3 Root 0.021 9.690 mg/g DE CH O CH O N Vindolidine:3 R3 = H N CH H3C H 3 H Vindoline: R = OCH3 N OH HO N CH O CH3O Vindolicine Leaf 0.07% H C 3 CH [27] MeO C H3 H 3CO Me 2 H CH O H 2 Vindolicine Leaf 0.07% HO N CHN3O 3 N N OH [27] HHC CHH 3CO Me MeO2C O3 H H O 2 HO N N OH N CH3O CH3O N VindolicineVindolicine LeafLeaf 0.07% 0.07% H C CH3 [27] [27] MeO C 3 CO Me 2 OH H H HO 2 N N N N OH HO O Vindolicine Leaf 0.07% OH CHH [27] H3C O3CO Me MeOHO2C O H H OH 2 Vindolicine Leaf 0.07% N N N N [27] O O CO Me MeO2C H H H H 2 O N N O HO OH Vindolicine Leaf 0.07% O O [27] O CO Me MeO2C O H H 2 N N N O O O O O N O H Vindolinine Leaf 0.02% N [27] O O H N VindolinineVindolinine LeafLeaf 0.02% 0.02% N [27] [27] CHH Vindolinine Leaf 0.02% N 3 [27] CO2Me NCHH3 Vindolinine Leaf 0.02% N CO2Me [27] H CH3 Vindolinine Leaf 0.02% N CO Me [27] 2 N H CH 3 Vindolinine Leaf 0.02% N CO2Me [27] CH3 N WholeWhole N CO2MeH Catharoseumine 0.7860.786 mg mg/kg/kg O O [28] [28] CH3 plantWholeplant N CO2MeH Catharoseumine 0.786 mg/kg O O [28] Wholeplant N CO2Me NH H Catharoseumine 0.786 mg/kg O O [28] plant N CO2Me Whole NH H Catharoseumine 0.786 mg/kg O [28] N COO Me Wholeplant 2 H Catharoseumine 0.786 mg/kg NH [28] plant O O NN CO2Me Whole H H Catharoseumine 0.786 mg/kg O [28] N COO2Me Hairyplant NH Tabersonine NR H [26] root Hairy NN CO2Me Tabersonine NR H H [26] root N CO2Me Hairy H Tabersonine NR N H [26] root N CO2Me Hairy NH Tabersonine NR H [26] N CO2Me Hairyroot H Tabersonine NR N H [26] Hairy root N CONH2Me2 Tryptamine Hairy NR H [26] Tabersonine NR H [26] Hairyroot N CO2Me root H NH2 Tryptamine NR N [26] Hairyroot H N CONH2Me2 Tryptamine NR HN [26] Hairyroot H NH2 Tryptamine NR N [26] Hairyroot H NH Tryptamine NR 2 [26] N root H Hairy NH Tryptamine NR N 2 [26] root H N H

Technologies 2020, 8, x FOR PEER REVIEW 5 of 17 Technologies 2020, 8, x FOR PEER REVIEW 5 of 17 Plant Alkaloids Quantity Chemical Structure Ref. PlantPart Alkaloids Quantity Chemical Structure Ref. Part

N − O Root 4.927 49.851 mg/g DE N H − O Root 4.927 49.851 mg/g DE N H MeO2C MeO2C

N [25,26 Catharanthine Leaf 0.2843 ± 0.0132 mg/g N [25,26] Catharanthine Leaf 0.2843 ± 0.0132 mg/g N CO2Me ] NCH3 CO2Me CH3 Vindolidine Leaf 0.14% Vindolidine Leaf 5.301−19.463 0.14% mg/g DE N Leaf 5.301−19.463 mg/g DE O − N O Stem 0.144 3.344 mg/g DE H O − [24,27 Stem 0.144 3.344 mg/g DE H O CO2Me [24,27] Vindoline R N H OH CO2Me Vindoline NCH H OH ] Root 0.021−9.690 mg/g DE R 3 Vindolidine: R = H − CH3 Root 0.021 9.690 mg/g DE Vindoline: R = OCH3 Vindolidine: R = H Vindoline: R = OCH3

CH O CH3O 3

CH3O CH3O CH H3C 3 N N H C CH3 H3 H Vindolicine Leaf 0.07% HO N N OH [27] H H CO Me MeO2C H H 2 Vindolicine Leaf 0.07% HO N N OH [27] CO Me MeO2C H H 2 O N N O

O O O O O O

N H Vindolinine Leaf 0.02% [27] H Vindolinine Leaf 0.02% N [27]

NCH3 CO2Me CH3 CO2Me

N Technologies 2020, 8, 80 5 of 16 Whole N H Catharoseumine 0.786 mg/kg O O [28] Wholeplant H Catharoseumine 0.786 mg/kg O O [28] plant N CO2Me Table 2. Cont. H N CO2Me Technologies 2020, 8, x FOR PEER REVIEW H 6 of 17

Alkaloids Plant Part Quantity Chemical Structure References Technologies 2020, 8, x FOR PEER REVIEW 6 of 17 Plant N TechnologiesAlkaloids 2020, 8 , x FOR PEER REVIEW Quantity Chemical Structure 6Ref. of 17 HairyPart N TechnologiesTabersonine 2020, 8, x FOR PEERPlant REVIEW NR H 6[26] of 17 Alkaloids Hairyroot Quantity Chemical Structure Ref. TechnologiesTabersonineTabersonine 2020, 8, x FOR PEERPlantPart Hairy REVIEW root NR NR H 6[26] of 17 [26] root N CO2Me Alkaloids Quantity ChemicalH Structure Ref. PlantPart Technologies 2020, 8, x FOR PEER REVIEW N CO2Me 6 of 17 Alkaloids Quantity ChemicalH Structure Ref. PlantPart N

CatharanthamineAlkaloids Leaf Quantity NR Chemical Structure Ref.[29] Part N Plant N Hairy NH Alkaloids Quantity Chemical StructureCO2Me 2 Ref. Tryptamine Part NR CH N [26] CatharanthamineTryptamine HairyLeafroot Hairy root NR NR 3 [29] [26] Tryptamine NR N NH2 [26] Catharanthamine Leaf NR N CON2Me [29] root H NCH3 Catharanthamine Leaf NR N CON2Me [29] CH H N 3 N CO2Me Catharanthamine Leaf NR N [29] CHH3 OH N CO Me CatharanthamineCatharanthamine Leaf Leaf NR NR N 2 N H [29] [29] 14′,15′- CH3 Whole NH OH N N CO2Me Didehydrocyclovin 0.071 mg/g DE CH H [30] H 3 OH N HH 14′,15′- plant OAc blastine Whole MeO2C N N H OH N H H Didehydrocyclovin14′,15′- 0.071 mg/g DE H [30] N HO CO2Me Wholeplant MeON H OAc NMeO2C N H Didehydrocyclovinblastine14′,15′- 0.071 mg/g DE H OH H [30] CH3 Wholeplant N N COOAcMe MeO2CMeO N HHO 2H Didehydrocyclovinblastine14′,15′- 0.071 mg/g DE H OH N H [30] plant CH OAc 14 ,15 -Didehydr Whole MeO C N 3HO CO2Me 0 blastine0 N 2MeO N H H Didehydrocyclovin1417-′,15′- Whole plant0.071 0.071 mg/g mg/ gDE DE H [30] [30] ocyclovinblastine CH CO Me Wholeplant MeO R N 3HO 2OAc DeacetoxycyclovinbDidehydrocyclovinblastine 0.0890.071 mg/g DE NMeO2C 1 H [30] 17- N CH H plant H R N 3 CO OAcMe MeO2MeOC 2 HO 2 blastinelastine Whole R1 Deacetoxycyclovinb17- 0.089 mg/g DE R3 N N CHH3 plant N HO CO2Me Whole MeOH HRR1 Deacetoxycyclovinblastine17- 0.089 mg/g DE H 2 N N R CH3 Wholeplant R 3 H H H R12 N H Deacetoxycyclovinblastine 0.089 mg/g DE H R [30] 17- MeO C N H R 4 Wholeplant N 2 H R 3 N H lastine HR12 H Deacetoxycyclovinb Whole 0.089 mg/g DE H H 17- N R3 N HO CO2Me Cycloleurosine plant 0.116 mg/g DE NMeO MeOC N H R4 [30] Whole 2 H HR12 H Deacetoxycyclovinblastine plant 0.089 mg/g DE H H 17-Deacetoxycy Whole N CH3 N R3 CORMe4 [30] Cycloleurosine Wholeplant plant0.116 0.089 mg/g mg/ gDE DE MeO2CMeO H R NN HHO 2H clovinblastinelastine 17-Deacetoxycyclovinblastine:H R12 = Et, RH2 = OH, R3 = R4 = H Wholeplant Cycloleurosine: R = R = -O-,H R = Et, R = OAc R [30] [30] MeO C1 3 2R3 NCH4 CO Me4 Cycloleurosine 0.116 mg/g DE N 2MeO N 3HOH 2H H H Wholeplant 17-Deacetoxycyclovinblastine:H R1 = Et, R2 = OH, R3 = R4 = H NCH3HO CO2RMe4 [30] Cycloleurosine 0.116 mg/g DE Cycloleurosine:NMeO2 CMeOR1 = R3 = -O-, R2 = Et, R4 = OAc H plant 17-Deacetoxycyclovinblastine: R1 = Et, RH2 = OH, R3 = R4 = H 17- Whole CH3 R [30] Cycloleurosine:MeO CR = R = -O-, R = Et,N R HO= OAcCO2Me4 Cycloleurosine Whole 0.116 mg/g DE 2MeO1 CHO3 2 4 Deacetoxyvinamidi Wholeplant 0.107 mg/g DE 17-Deacetoxycyclovinblastine:O R1 = Et, R2 = OH, R3 = R4 = H Cycloleurosine: R1 = R3 = -O-, R2 = Et,CH R34 = OAcCO Me CycloleurosineCycloleurosine17- plant Whole plant0.116 0.116 mg/g mg/ gDE DE MeON N HO 2 Whole CHO ne plant 17-Deacetoxycyclovinblastine: R1 = Et, R2 = OH, R3 = R4 = H Deacetoxyvinamidi 0.107 mg/g DE O CH 17- Cycloleurosine: R1 = R3 = -O-, R2 = Et, R43 = OAc Wholeplant CHON N H 17-Deacetoxycyclovinblastine: R1 = Et, R2 = OH, R3 = R4 = H Deacetoxyvinamidi17-ne 0.107 mg/g DE O H Cycloleurosine: R1 = R3 = -O-, R2 = Et, R4 = OAc Wholeplant N NCHO H Deacetoxyvinamidi17-ne 0.107 mg/g DE H O N HH [30] plant N H R Whole MeO2C CHO ne N N H H Deacetoxyvinamidi17-Deaceto17- Whole 0.107 mg/g DE H O H N H N HO CO2Me [30] Vinamidine WholeplantWhole plant0.071 0.107 mg/g mg/ gDE DE N MeOCHO N H R MeO2C H Deacetoxyvinamidixyvinamidinene plant 0.107 mg/g DE H O H Whole H CH3 [30] plant N N CORMe [30] Vinamidine 0.071 mg/g DE MeO2CMeO NN HHO 2H ne H17-Deacetoxyvinamidine: R = HH Wholeplant H R [30] Vinamidine:MeO C R = OAc NCH CO Me Vinamidine 0.071 mg/g DE N 2MeO N 3HOH 2H H H Wholeplant 17-Deacetoxyvinamidine:H R = H [30] NCH3HO CO2RMe Vinamidine 0.071 mg/g DE NMeOVinamidine:2CMeO R = OAc H plant H H Whole 17-Deacetoxyvinamidine: RCH = H [30] N 3 CO RMe VinamidineLeurosineVinamidine Whole plant0.1340.071 0.071 mg/g mg/ gDE DE Vinamidine:MeO2MeOC R = O ARc HO 2 Wholeplant 17-Deacetoxyvinamidine:1 R = H N CH3 CO Me Vinamidine Whole 0.071 mg/g DE Vinamidine:MeO R = O Ac N HO 2 Leurosine 0.134 mg/g DE R2 plant 17-Deacetoxyvinamidine:R1 R = H Wholeplant R CH3 Leurosine 0.134 mg/g DE Vinamidine:N R = OAc 3 N H HRR1 Wholeplant 17-Deacetoxyvinamidine:H 2 R = H NVinamidine:N R = OAcR Leurosine 0.134 mg/g DE R13 H H plant R2 N H Whole N H H R OAc [30] Leurosine 0.134 mg/g DE MeO2C R 3 H LeurosineWhole Whole plant 0.134 mg/g DE N R12 N H Wholeplant H H Leurosidine 0.107 mg/g DE N H R CO Me Leurosine plant 0.134 mg/g DE N MeO 3 NN HHO 2OAc [30] MeO2C HR12 H [30] Wholeplant H H Leurosidine 0.107 mg/g DE N R CH3 OAc [30] NMeO C 3 N H CO HMe 2 MeO R2 N HO 2 Wholeplant Leurosine: R = R = -O-,H R = Et H 1 3 H 2 OAc [30] Leurosidine 0.107 mg/g DE Leurosidine:MeO C R = OH, R R =3 Et, R = H CO Me Wholeplant N 2MeO 1 2 NNCH33HOH 2H H H Leurosidine 0.107 mg/g DE Leurosine: R = R =H -O-, R = Et N 1 3 2 NCH HO CO2OAcMe [30] plant Leurosidine:MeO2CMeO R = OH, R = Et, R3 = H H LeurosidineWhole Whole plant 0.107 mg/g DE 1 2 3H Leurosine: R = R = -O-, R = Et Leurosidine 0.107 mg/g DE 1 3 2 CH3 OAc [30] MeO C N HO CO2Me Wholeplant Leurosidine:2MeO R1 = OH, R2 = Et, R3 = H Leurosine: R = R = -O-, R = Et Leurosidine 0.107 mg/g DE 1N 3 2 NCH3 CO Me plant Leurosidine:MeO R1 = OH, R2 = Et, R3 =HO H 2 Leurosine: R CHO = R = -O-, R = Et 1 3 2 CH N N 3H Leurosidine: R1 = OH,O R2 = Et, R3 = H Leurosine: R = R = -O-, R = Et Whole 1NCHO3 2 Leurosidine:N R = OH, R = Et, R = H H [30] CatharineCatharine Whole plant0.098 0.098 mg/g mg/ gDE DE H 1 2 N 3HH [30] plant O MeO C NCHO OAc Whole N 2 N H O H Catharine 0.098 mg/g DE H CHO H [30] Wholeplant N N HO CO2Me NMeO MeOC N H OAc Catharine 0.098 mg/g DE H 2 O H [30] CHO CH H Wholeplant N N 3 MeO2C NN HHO COOAc2HMe Catharine 0.098 mg/g DE H MeO O H [30] plant CHO Whole MeO C NCH COOAcMe N 2MeO N 3HOH 2H Catharine 0.098 mg/g DE H O H [30] Wholeplant NCH3HO CO2Me NMeO2CMeO OAcH Catharine 0.098 mg/g DE H H [30] CH plant N 3 CO Me MeO2MeOC HO OAc2

CH3 CO Me MeO N HO 2

CH3

Technologies 2020, 8, 80 6 of 16

Technologies 2020, 8, x FOR PEER REVIEW 7 of 17

Table 2. Cont. Plant Alkaloids Quantity Chemical Structure Ref. Part Alkaloids Plant Part Quantity Chemical Structure References

N H Whole H CathachunineCathachunine Whole plant0.223 0.223 mg/g mg/ gDE DE N [31] [31] plant H H

MeO2C

CO Me MeO N HO 2 CH3

DE: Dry extract. NR: Not reported. DE: Dry extract. NR: Not reported.

Besides well-known well-known alkaloids, alkaloids, new new phenolic phenolic compounds compounds were also were found also in found the C. inroseus the leaf,C. roseus leaf, stem,stem, seed and and petal petal (Table (Table 3).3 ).

Table 3. Identified phenolic compounds in C. roseus. Table 3. Identified phenolic compounds in C. roseus. Quantity Plant Parts Phenolic Compounds Quantity Reference Plant Parts Phenolic Compounds (mg/kg Dry Basis) References 3-O-caffeoylquinic acid (mg 769.9/kg Dry ± 12.7 Basis) [32,33] 4-O3--caffeoylquinicO-caffeoylquinic acid acid 2874.6 769.9 ± 151.612.7 [32,33] [32 ,33] ± 5-O4--caffeoylquinicO-caffeoylquinic acid acid 2874.6 22.5 ± 1.5151.6 [32,33] [32 ,33] Stem ± Quercetin-3-O-(2,6-di-5-O-caffOeoylquinic-rhamnosyl-galactoside) acid 190.5 22.5 ± 1.53.1 [32,33] [32 ,33] Stem ± Quercetin-3-O-(2,6-di-O-rhamnosyl-galactoside) 190.5 ± 3.1 [32,33] Kaempferol-3-O-(2,6-di-O-rhamnosyl-galactoside) 190.8 ± 5.3 [32,33] Kaempferol-3-O-(2,6-di-O-rhamnosyl-galactoside) 190.8± 5.3 [32,33] Isorhamnetin-3-O-(2,6-di-O-rhamnosyl galactoside) 78.6 ± 3.9 [32,33] Isorhamnetin-3-O-(2,6-di-O-rhamnosyl galactoside) 78.6 3.9 [32,33] 3-O-caffeoylquinic acid 2971.6± ± 15.6 [32,33] Kaempferol-3-O-(2,6-di-3-OO-rhamnosyl-galactoside)-7--caffeoylquinic acidO-hexoside 2971.652.7 ± 1.015.6 [32,33] [32 ,33] ± Kaempferol-3-O-(2,6-di-O-rhamnosyl-galactoside)-7-O-hexoside 52.7 ± 1.0 [32,33] 4-O-caffeoylquinic acid 5156.8 ± 137.2 [32,33] Leaf 4-O-caffeoylquinic acid 5156.8 ±137.2 [32,33] Leaf 5-O-caffeoylquinic acid 187.7± 0.5 [32,33] 5-O-caffeoylquinic acid 187.7 ± 0.5 [32,33] Quercetin-3-O-(2,6-di-O-rhamnosyl-galactoside) 310.9 ± 5.0 [32,33] Quercetin-3-O-(2,6-di-O-rhamnosyl-galactoside) 310.9 5.0 [32,33] Kaempferol-3-O-(2,6-di-O-rhamnosyl-galactoside) 8.5 ±± 5.3 [32,33] Kaempferol-3-O-(2,6-di-O-rhamnosyl-galactoside) 8.5 5.3 [32,33] Kaempferol-3-O-(2,6-di-O-rhamnosyl-galactoside)-7-O-hexoside 292.3± ± 0.3 [32,33] Kaempferol-3-O-(2,6-di-O-rhamnosyl-galactoside)-7-O-hexoside 292.3 ± 0.3 [32,33] Quercetin-3-O-(2,6-di-O-rhamnosyl-galactoside) 582.7 ± 6.6 [32,33] Quercetin-3-O-(2,6-di-O-rhamnosyl-galactoside) 582.7 ±6.6 [32,33] Kaempferol-3-O-(2,6-di-O-rhamnosyl-galactoside) 2714.2± 4.3 [32,33] Kaempferol-3-O-(2,6-di-O-rhamnosyl-galactoside) 2714.2 4.3 [32,33] Seeds Kaempferol-3-O-(2,6-di-O-rhamnosyl-glucoside) 56.6 ±± 0.4 [32,33] Seeds Kaempferol-3-O-(2,6-di-O-rhamnosyl-glucoside) 56.6 0.4 [32,33] Isorhamnetin-3-O-(2,6-di-O-rhamnosyl-glucoside) 354.1± ± 8.2 [32,33] Isorhamnetin-3-O-(2,6-di-O-rhamnosyl-glucoside) 354.1 8.2 [32,33] Kaempferol-3-O-(6-O-rhamnosyl-galactoside) 112.1 ±± 16.0 [32,33] Kaempferol-3-O-(6-O-rhamnosyl-galactoside) 112.1 16.0 [32,33] ± Isorhamnetin-3-Isorhamnetin-3-O-(6-O-(6-O-rhamnosyl-glucoside)O-rhamnosyl-glucoside) 372.0 65.2 [32,33] [32 ,33] 4-O-caffeoylquinic acid 11153.2± ± 126.4 [32,33] 4-O-caffeoylquinic acid 11153.2 126.4 [32,33] Quercetin-3-O-(2,6-di-O-rhamnosyl-galactoside) 1027.9± ± 7.0 [32] Quercetin-3-O-(2,6-di-O-rhamnosyl-galactoside) 1027.9 7.0 [32] Kaempferol-3-O-(2,6-di-O-rhamnosyl-galactoside) 8120.8 ±± 74.4 [32] Kaempferol-3-O-(2,6-di-O-rhamnosyl-galactoside) 8120.8 74.4 [32] Kaempferol-3-O-(2,6-di-O-rhamnosyl-glucoside) 4296.3 ± 34.4 [32] Petal Kaempferol-3-O-(2,6-di-O-rhamnosyl-glucoside) 4296.3 34.4 [32] Petal ± Kaempferol-3-Kaempferol-3-O-(6-O-(6-O-rhamnosyl-galactoside)O-rhamnosyl-galactoside) 9567.2 98.5 [32,33] [32 ,33] ± Kaempferol-3-Kaempferol-3-O-(6-O-(6-O-rhamnosyl-glucoside)O-rhamnosyl-glucoside) 4639.8 21.9 [32,33] [32 ,33] ± Isorhamnetin-3-Isorhamnetin-3-O-(6-O-(6-O-rhamnosyl-galactoside)O-rhamnosyl-galactoside) 989.2 ± 33.0 [32,33] [32 ,33] ± Isorhamnetin-3-Isorhamnetin-3-O-(6-O-(6-O-rhamnosyl-glucoside)O-rhamnosyl-glucoside) 1330.4 ± 10.8 [32,33] [32 ,33] ± This plant has been found to possess a number of important bioactive components that greatly contributeThis plantto the hasherbal been medicine found industry; to possess however, a number their amounts of important present bioactive in the plant components are often low. that greatly contributeOf note, the tobiosynthesis the herbal of medicine plant secondary industry; metaboli however,tes is affected their amounts by the biotic present and abiotic in the factors. plant are often low.Particularly, Of note, environmental the biosynthesis stresses of planthave been secondary found metabolitesto stimulate the is a productionffected by theof secondary biotic and abiotic factors.metabolites Particularly, including environmentalalkaloids because stresses of the plant have protective been found function. to stimulate Hence, themore production in vitro and of in secondary vivo studies focus on increasing the amounts of these compounds through changing environmental metabolites including alkaloids because of the plant protective function. Hence, more in vitro and conditions such as light, salinity, soil types and nutrients, drought and metal stress. In particular, inMisra vivo andstudies Gupta focus [34] found on increasing that salinity the (100 amounts mM NaCl) of these stimulated compounds indole throughalkaloid changingaccumulation environmental in conditionsthe C. roseus such leaves as and light, roots. salinity, The amount soil types of ajmalicine and nutrients, in C. roseus drought increased and with metal the treatment stress. In of particular, Misracadmium and (concentration Gupta [34] found ranging that from salinity 0.05 to (100 0.4 mM) mM from NaCl) 24 stimulatedto 48 h and excretion indole alkaloid into the accumulationculture in themedium,C. roseus especiallyleaves for and cells roots. at the The mid-exponential amount of ajmalicine growth phase in C.[35]. roseus The studyincreased of Binder with et the al. [36] treatment of cadmiumindicated that (concentration UV-B light stimulated ranging fromalkaloid 0.05 production to 0.4 mM) in C. from roseus 24 hairy to 48 root h and due excretionto protein intokinase the culture medium, especially for cells at the mid-exponential growth phase [35]. The study of Binder et al. [36] indicated that UV-B light stimulated alkaloid production in C. roseus hairy root due to protein kinase stimulation. Additionally, biomass and alkaloid yield in cell suspension culture of C. roseus was Technologies 2020, 8, 80 7 of 16 found to increase along with the increased total nitrogen and phosphate at an adequate pH of test culture medium because nitrogen is an important component of alkaloids, while phosphorus affects the synthesis of alkaloids [37]. A similar phenomenon was also found in other medicinal plants. Oueslati et al. [38] found that polyphenol content of the Mentha pulegium leaf irrigated with a nutrient solution containing 100 mM NaCl for 14 days increased more than 3-fold higher than that of the control. The levels of terpenes increased in the green leaves of the thyme plants in response to drought stress [39].

4. Preparation and Recovery of Bioactive Compounds from C. roseus

4.1. Preparation of Dried C. roseus In previous studies, various drying methods, such as freeze drying, air drying, low-temperature drying, infrared drying or drying under the shade have been applied to prepare dried C. roseus for further recovery of bioactive compounds (Table5). The results showed that drying methods significantly affected the retention of bioactive compounds in C. roseus material because bioactive compounds are sensitive to heat, light and oxygen [40]. Freeze drying has been reported as a prominent and effective drying method in terms of the retention of bioactive compounds, but it is costly and not typically available in some of the regions where the plant is collected and processed [5]. Among thermal drying methods, infrared drying at 35 ◦C was found to be suitable for saponin retention within the C. roseus stem and root, while vacuum drying at 50 ◦C was suggested for drying the leaf and the flower, which contained high levels of phenolics and flavonoids [40]. However, there is no report on the effect of drying conditions on the individual bioactive compounds present in this plant material, suggesting future studies on investigating the optimum drying conditions for recovering target compounds from C. roseus are required.

Table 4. Preparation and recovery of bioactive compounds from C. roseus.

Plant Part Compounds Procedures References Freeze-drying sample, Extracting sample (200 250 mg) with 80 mL of methanol − for 3 h in a Soxhlet extraction apparatus (reﬂux rate of 12 15 siphons/h), Vindoline, ajmalicine, serpentine, − Hairy root Evaporating and diluting with 1.5 volumes of a 5 mM [41] and catharanthine (NH4)2HPO4 solution, Fractionating using a 300-mg C18 Maxi-Clean cartridge, then eluting successively with 4 mL of mixture of MeOH:5 mM (NH4)2HPO4 (60:40, 95:5, and 100:0, v/v).

Drying the sample at 60 ◦C for 48 h and grinding the dried material, Extracting dried sample (5 g) overnight with 90% ethanol (30 mL) at room temperature (3 times), Filtering ethanol extract and vacuum concentrating at 40 ◦C, Redissolving the residue in ethanol (10 mL), then diluting with water (10 mL) and acidifying with 3% hydrochloric acid (10 mL), Washing with hexane (3 30 mL), × Cooling the aqueous portion down to 10 C, adjusting pH 8.5 Vindoline, catharanthine, ◦ Leaf with ammonium, [42] vincristine and vinblastine Extracting with chloroform (3 30 mL), × Washing with water and evaporating chloroform to get the dried residue, Redissolving in 1 mL chloroform, Separating with a silica Sep-Pak cartridge (Waters) pre-saturated with chloroform, Washing with 5 mL each of chloroform and then with chloroform: methanol (9:1, v/v) before drying over anhydrous sodium sulphate, Evaporating to dryness. Serpentine, vincristine, vindoline, Freeze-drying and grinding the sample, catharanthine, vinblastine, Ultrasound-assisted extracting at room temperature (RT) in Hairy root [26] tabersonine, tryptamine and 1 mL of MeOH for 60 min, ajmalicine Centrifuging and ﬁltering to get the extract solution. Technologies 2020, 8, 80 8 of 16

Table 5. Preparation and recovery of bioactive compounds from C. roseus.

Plant Part Compounds Procedures References Drying and grinding the plant material, Extracting dried sample with 95% EtOH, subsequently evaporating to get a residue, 14 ,15 -didehydrocyclovinblastine, 0 0 Redissolving with water and then extraction with CHCl , 17-deacetoxycyclovinblastine, 3 Separating CHCl fraction using silica gel column 17-deacetoxyvinamidine, 3 Whole plant chromatography (200–300 mesh, Qingdao Marine Chemical [30,31] vinamidin, leurosine, catharine, Factory, Qingdao, China), then using ODS column (YMC, cycloleurosine, leurosidine and Kyoto, Japan) to get subfractions, cathachunine Purifying subfractions by preparative HPLC on a COSMOSIL C18 preparative column (5 µm, 20 250 mm, × Nacalai Tesque, Kyoto, Japan). Air-drying and grinding the plant material, Extracting dried sample with MeOH under reﬂux, subsequently evaporating to get a residue, Redissolving with water and adjusting to pH 3 with tartaric acid, Defatting by petroleum ether, and then subjecting to cation ion exchange resins, Whole plant Catharoseumine Washing the resins by water and basiﬁed, then eluting with [28] EtOAc and MeOH, The EtOAc fraction was subjected to silica gel column chromatography eluting with CHCl /CH OH (100:1 1:1) to 3 3 → give 5 fractions (A–E). Fraction A was further applied to column chromatography over reverse-phase C-18 silica gel, Sephadex LH-20, and silica gel chromatography to get catharoseumine.

Drying the leaf at 40 ◦C and grinding dried sample, Defatting dried sample (1 kg) using n-hexane (10 L) for 3 days at RT, then removing n-hexane to get the residue, The residue was first wetted with 25% ammonia for 1 h, followed by soaking twice with dichloromethane (10 L) for 3 days at RT, Filtering and drying dichloromethane extract under reduced pressure, Acid-base extracting dichloromethane extract using 5% Leaf Vindogentianine hydrochloric acid and 25% ammonia solution to get an [43] alkaloid crude extract, Separating alkaloid crude extract using silica column chromatography (CC, diameter: 10cm; Merck, Kenilworth, NJ, USA), then flushing using a solvent mixture of CH2Cl2 and MeOH (a ratio of 40:60, 20:80 (v/v)) before 100% MeOH to get fractions (1–15). Applying fraction 4 to preparative thin layer chromatography silica gel F254 (1 mm; Merck) under ammonia vapour to get vindogentianine. Drying the samples under shade and grinding dried sample, Ultrasonic extraction with ethanol for 30 min at 30 ◦C (sample-to-solvent ratio of 1/10, g/mL) (53 KHz, Bandelin Ajmaline, yohimbine, vindesine, SONOREX, Berlin) and then kept at RT for 24 h, Leaf, stem and ajmalicine, serpentine, vincristine, The extracts were filtered through filter paper (Whatman [24] root vinblastine, vindoline, vinpocetine No. 1) and filtrates were collected. The residues were and reserpine re-extracted with fresh solvent again. The combined filtrates were evaporated to dryness under reduced pressure. Freeze-drying sample, Ultrasound-assisted extracting using methanol:water (1:1) as Caffeoylquinic acids, quercetin, the extraction solvent for 1 h, macerating for 15 h and Leaf, stem, seed kaempferol, isorhamnetin and ultrasonicating again for 1 h (leaf or stem/solvent: 1/10 g/mL, [32] and petal their derivatives petal/solvent: 1/100 g/mL, seed/solvent: 1/3 g/mL), Centrifuging, filtering and then lyophilising to obtain the powdered phenolic extract. Freeze-drying sample, Caffeoylquinic acids, quercetin, Extraction with boiling water for 20 min (sample-to-solvent Leaf, stem, seed kaempferol, isorhamnetin and ratio of 1/200 g/mL), [33] and petal their derivatives Filtering and then lyophilising to obtain the powdered phenolic extract. MeOH: Methanol. EtOH: Ethanol. EtOAc: Ethyl acetate. RT: Room temperature. Technologies 2020, 8, 80 9 of 16

4.2. Recovery of Bioactive Compounds from C. roseus There have been numerous extraction techniques used for the recovery of bioactive compounds from C. roseus, including conventional extraction, ultrasonic-assisted extraction and Soxhlet extraction (Table5) using the common solvents methanol and ethanol. Soaking materials with 90% ethanol and leaving the mixture overnight has been used to extract vindoline, catharanthine, vincristine and vinblastine from C. roseus leaf (Table5)[ 42]. To obtain the phenolic extract, lyophilized and powdered plant material (leaves, stems, seeds and petals) were extracted with boiling water for 20 min [33]. In this method, the solvent diffuses into the material matrix, dissolves solutes and liberates them from the plant matrix. This is driven by a concentration gradient at the solid-liquid interphase that exists until the equilibrium is established [44]. This method is cheap and simple but requires prolonged extraction times that may lead to the loss of active compounds through oxidation, ionization and hydrolysis. The Soxhlet extraction was used to prepare the C. roseus hairy root extract for isolation of vindoline, ajmalicine, serpentine and catharanthine [41]. Although this method is simple, well established and inexpensive, it is also time consuming and requires large quantities of solvent. Further, it occurs at the boiling point of solvents over a long period that may cause the thermal degradation of bioactive compounds [45]. Ultrasonic extraction is an emerging extraction technique with numerous advantages over conventional thermal extraction methods including shorter extraction times, higher extraction yields, energy and solvent cost savings. C. roseus has been subjected to ultrasonic extraction to obtain specific target compounds (Table5)[ 24,26,32,40,46]. Other advanced extraction techniques such as microwave-assisted extraction (MAE), enzyme-assisted extraction, pulsed electric field extraction, pressurised liquid extraction and supercritical fluid extraction, which can also reduce extraction time, solvent consumption and environmental pollution could be considered; however, industrial up-scaling potential needs to be taken into consideration. To obtain phenolic extracts from C. roseus, Ferreres et al. [32] and Pereira et al. [33] applied different extraction methods that are ultrasound-assisted extraction and maceration using methanol:water (1/1) as the extraction solvent at room temperature for 17 h versus extraction with boiling water for 20 min (Table5). In the latter method, water—a cheap, accessible and environmentally friendly solvent—was used, and shorter extraction time was applied as the extraction process was conducted at the boiling point, while the 50% aqueous methanol was used in the former one. However, the phenolic extraction efficiency of these methods could not be fully clarified as the quantity of individual phenolic compounds has not been determined in the former study. C. roseus crude extracts could be further separated using chloroform or using ion exchange resins to collect fractions that would be subjected to silica gel column chromatography to collect subfractions, then the individual compounds further purified using column chromatography over Sephadex LH-20 and preparative TLC or preparative HPLC [28,30,31,42]. The detailed recovery process of bioactive compounds from C. roseus is presented in Table5. Of note, besides extraction techniques various extraction parameters, such as temperature, time, solvents, solvent-to-sample ratio, particle size, solvent pH and pressure have been found to significantly affect extraction efficiency and stability of bioactive compounds from plant materials [40,46–50], future studies are recommended to investigate the optimal extraction conditions for recovery of groups or individual alkaloids and/or phenolic compounds from C. roseus for further extraction and isolation. In addition, advanced techniques should be applied to identify and test the potential use of unknown compounds in C. roseus.

5. Potential Use for Health Beneﬁts

5.1. Potential Use as an Anticancer Agent C. roseus has been found to contain a range of alkaloids possessing anticancer activity including vinblastine, vincristine, vindoline, vindolidine, vindolicine, vindolinine and vindogentianine [27,43,51]. They inhibit cell proliferation through changing the microtubular dynamics, which induces Technologies 2020, 8, 80 10 of 16 apoptosis [52]. Of these, vinblastine and vincristine were the first plant-derived anticancer agents deployed for clinical use [51]. Vinblastine sulphate has been utilised for treatment of Hodgkin’s disease, lymphosarcoma, choriocarcinoma, neuroblastoma, carcinoma of breast and lungs and other organs in acute and chronic leukaemia. Vincristine sulphate, an oxidised form of vinblastine [52] arrests mitosis in the metaphase and is effective in treating acute leukaemia (in children), lymphocytic leukaemia, Hodgkin’s disease, Wilkins’s tumour, neuroblastoma and reticulum cell sarcoma [2]. A range of other indole alkaloids extracted from C. roseus has been found to exhibit potent cytotoxic activity against various cancer cell lines. Catharoseumine, a new monoterpenoid indole alkaloid isolated from the C. roseus whole plant was found to possess an inhibitory effect on the human promyelocytic leukaemia HL-60 cell line with an IC50 of 6.28 µM[28]. Moreover, three newly isolated dimeric indole alkaloids including 140,150-didehydrocyclovinblastine, 17-deacetoxycyclovinblastine, 17-deacetoxyvinamidine and five known compounds (vinamidine, leurosine, catharine, cycloleurosine and leurosidine) possessed in vitro inhibition of cell viability against the human breast cancer cell line MDA-MB-231 with an IC50 range of 0.73–10.67 µM[30]. Importantly, cathachunine, a new bisindole alkaloid from C. roseus, displayed antitumour effects on human leukaemia cells, but at much lower cytotoxicity against normal human endothelial cells, indicating that its activity inhibited selectively toward leukaemia cells. Along with the studies on anticancer activity of individual alkaloids from C. roseus, effect of the entire crude extract on various cancer cell lines was also investigated. Recent findings found that C. roseus root and stem extract possessed strong in vitro cytotoxic activity against a panel of cancer cell lines [53,54]. Similarly, the study of Fernández-Pérez et al. [55] confirmed that the powerful antitumor activity of the indole alkaloid-enriched extract obtained from C. roseus cell cultures did not result from the effect of a single compound, but depended on the joint action of bioactive compounds. These results revealed the synergistic effect and positive interaction of the bioactive components present in C. roseus against cancer cells, which has also been found in other plant materials [56,57] and considered as a strategy for cancer treatment [58]. This phenomenon is possible due to the assistance of compounds with little or no activity to the main active compounds through helping the active component reach the target by improving bioavailability, decreasing metabolism and excretion, complementing mechanisms of action, reversal of resistance and modulating side effects [59].

5.2. Potential Use as an Antidiabetic Agent C. roseus has been traditionally used as a treatment for diabetes in many regions of the world (Table1). Juice from the leaf of C. roseus was reported to produce a dose-dependent reduction in blood glucose of both normal and diabetic rabbits [60]. The whole plant C. roseus methanolic extract displayed eﬀective antihyperglycaemic activity, correlating with improvement in body weight, lipid proﬁle and regeneration of β-cells of the pancreas in diabetic rats [61]. Recently, Tiong et al. [27] investigated the in vitro antidiabetic properties of four pure alkaloids including vindoline, vindolidine, vindolicine and vindolinine isolated from C. roseus leaf via the assays of 2-NBDG glucose uptake and inhibition of PTP-1B, an enzyme that regulates negatively the insulin signalling pathway. Improving glucose uptake in pancreatic or muscle cells could amend the hyperglycaemic conditions of type 2 diabetes. It was found that the four alkaloids increased the glucose uptake in mouse β-TC6 pancreatic and mouse myoblast C2C12 cells, coupled with their inhibition of PTP-1B. Among them, vindolicine showed the highest activity. The results supported the traditional utilisation of C. roseus for diabetic treatment, highlighting that C. roseus is a potent source for further exploring antidiabetic agents.

5.3. Potential Use as Anti-Alzheimer’s Disease Agents Alzheimer’s disease (AD) is a neurodegenerative disorder of the central nervous system and accounts for 50–60% of dementia in patients [62]. It is characterised by profound memory impairment, emotional disturbance and personality changes in the late stages of life [63]. Cholinergic neurons in the neocortex and hippocampus are suggested to be predominantly affected in AD, resulting in the cholinergic Technologies 2020, 8, 80 11 of 16 hypothesis, which associates AD symptoms to cholinergic deficiency. Therefore, symptomatic treatment for AD has been focused upon augmenting brain cholinergic neurotransmission [64]. The current effective AD therapy is to increase acetylcholine (Ach) levels by inhibiting acetylcholinesterase (AchE), an enzyme responsible for degrading Ach in the synaptic cleft [62,64]. The aqueous extract of C. roseus leaf, stem and root has been shown to effectively inhibit AchE in an in vitro microassay [33,65]. Additionally, serpentine, an alkaloid present in C. roseus leaf, stem and root [24] displayed a strong activity against AchE with a low IC50 value (0.775 µM) [66]. These findings revealed that this plant is a potential source of active compounds for the pharmacological management of neurodegenerative conditions including Alzheimer’s disease.

5.4. Other Beneficial Effects Many microorganisms cause disease in plants, animals and humans, leading to the loss of crops, spoilage of food and increasing problems for human health [67]. Further, microbial resistance, which has become a rising challenging for agriculture and human health, requires more effective antimicrobial agents [52]. Ethanol extracts of C. roseus leaf, stem, flower and root have been reported to display antibacterial activity against various bacteria [68,69]. Additionally, the saponin-enriched fractions from C. roseus stem and root revealed their potent antifungal activity against Candida albicans and Aspergillus niger [53,54]. Moreover, individual compounds isolated from C. roseus were identified as potent antimicrobial agents. Yohimbine, an alkaloid isolated from C. roseus leaf, stem and root [24] was found to exhibit not only antibacterial, antifungal activities but also antiviral activity against herpes simplex virus (type 1) [70]. Remarkably, catharoseumine present in C. roseus whole plant was found to inhibit Plasmodium falciparum falcipain-2, a protozoan parasite that causes malaria, with an IC50 value of 4.06 µM[28]. Wound healing is a complex and dynamic process of restoring cellular structures and tissue layers in damaged tissue as closely as possible to its normal state. The ethanol extracts of C. roseus flower and leaf has been demonstrated to possess wound healing activity in rats [71,72], resulting from the astringent and antimicrobial properties of bioactive components present in the C. roseus extract [73]. In particular, the leaf extract promoted wound healing in diabetic rats [71]. The results coincided with the findings of Singh et al. [73] who found that the methanol extract of C. roseus leaf significantly increased wound contraction in streptozotocin induced diabetic mice. The wound healing activity of C. roseus extracts is promising to overcome the poor wound healing typical in the diabetic condition. In these instances, wounds commonly slow healing and can exist for weeks, raising significant challenges for wound care and control in clinical practice. The leaf extract of C. roseus was shown to possess hypotensive and lipid lowering effects in adrenaline-induced hypertensive rats [74]. Ajmalicine found naturally in C. roseus [24] has been considered as a hypotensive agent as it acted as an α-adrenergic receptor antagonist [52,75]. Additionally, C. roseus extracts and isolated alkaloids including vindoline, vindolidine, vindolicine and vindolinine were found to possess antioxidant properties [27,46,53,54], which could reduce and prevent the oxidation of other molecules. Although oxidation reactions are crucial for life, they produce free radicals and then start a cascade of biochemical reactions, causing oxidative stress that damage or kill cells and contribute to cell ageing, cardiovascular disease, brain disease (associated with Alzheimer0s disease), mutagenic changes and cancerous tumour growth [76,77]. Technologies 2020, 8, 80 12 of 16

6. Conclusions C. roseus can be considered as a rich source of alkaloids and phenolics, which possess diverse biological properties including anticancer, antidiabetic, antioxidant, antimicrobial and antihypertensive activities. Numerous alkaloids and phenolics have been identified in this material but many compounds are unknown. Consequently, the identification and isolation of new phytochemicals within the different structural components of C. roseus should be continued. In addition, potential uses of bioactive compounds derived from this material need to be further investigated for applications in the nutraceutical and pharmaceutical industries.

Author Contributions: Conceptualization, H.N.T.P., Q.V.V., M.C.B. and C.J.S.; Supervision, Q.V.V., M.C.B. and C.J.S.; Writing—original draft, H.N.T.P.; and Writing—review and editing, Q.V.V., M.C.B. and C.J.S. All authors have read and agreed to the published version of the manuscript. Funding: This work received no external funding. Acknowledgments: The authors thank the University of Newcastle, Australia for granting Hong Ngoc Thuy Pham the scholarships (UNIPRS and UNRSC 50:50) in 2015. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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