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. 2020 Sep 1;58(6):2054–2067. doi: 10.1007/s13197-020-04745-3

Extraction methods of butterfly pea (Clitoria ternatea) flower and biological activities of its phytochemicals

Ethel Jeyaseela Jeyaraj 1, Yau Yan Lim 1, Wee Sim Choo 1,✉
PMCID: PMC8076379  PMID: 33967304

Abstract

Clitoria ternatea or commonly known as ‘Butterfly pea’ has been used traditionally in Ayurvedic medicine in which various parts of the plants are used to treat health issues such as indigestion, constipation, arthritis, skin diseases, liver and intestinal problems. The flowers of C. ternatea are used worldwide as ornamental flowers and traditionally used as a food colorant. This paper reviews the recent advances in the extraction and biological activities of phytochemicals from C. ternatea flowers. The application of maceration or ultrasound assisted extraction greatly increased the yield (16–247% of increase) of phytochemicals from C. ternatea flowers. Various phytochemicals such as kaempferol, quercetin and myricetin glycosides as well as anthocyanins have been isolated from C. ternatea flowers. Clitoria ternatea flower extracts were found to possess antimicrobial, antioxidant, anti-inflammatory, cytotoxic and antidiabetic activities which are beneficial to human health. Clitoria ternatea flower is a promising candidate for functional food applications owing to its wide range of pharmacotherapeutic properties as well as its safety and effectiveness.

Keywords: Anthocyanin, Antibacterial, Anticancer, Anti-inflammatory, Antioxidant, Flavonol

Introduction

Aromatic and medicinal plants have been used for therapeutic, religious, cosmetic, nutritional, and beautification purposes since ancient times and humanity of all civilizations and culture are familiar with their usage (Senica et al. 2019; Senkal et al. 2019; Gecer et al. 2020). Clitoria ternatea plant is classified in the kingdom Plantae, phylum Tracheophyta, class of Magnoliopsida and a family of Fabaceae (Jamil et al. 2018). Clitoria ternatea is a perennial climber (2–3 m in height) and is known by its common name as butterfly pea or blue pea flower (Mukherjee et al. 2008). In other regions it is known as aparajita (Bengali), kajroti (India), cunha (Brazilian), cunhã, fula criqua (Portuguese), lan hu die (Chinese), bunga biru, tembang telang (Indonesian), bunga telang (Malaysian), clitoria azul (Spanish), dangchan (Thai), chi dậu biếc (Vietnamese) and mavi kelebek sarmaşığı (Turkish) (Kosai et al. 2015; Subramanian and Prathyusha 2011; Mukherjee et al. 2008). It is commonly grown as an ornamental plant and is also used as a revegetation species while in Southeast Asia, the blue flower pigment is traditionally used as food colorant (Havananda and Luengwilai 2019; Oguis et al. 2019) The plant is known to be suitable as a cover crop and green manure having the ability to not only suppress perennial weeds but able to enrich the soil by nitrogen fixation (Chauhan et al. 2012; Reid and Sinclair 1980). C. ternatea plant is widely distributed in India, Phillipines, in other tropical Asian countries, South and Central America, the Caribbean and Madagascar (Sivaranjan and Balachandran 1994; Ambasta 1988).

Clitoria ternatea is considered as a nootropic herb in Ayurvedic medicine (Chauhan et al. 2017). It grows well in full sunlight/partially shaded area in which seed germination takes about 1–2 weeks while 4 weeks is required for flowering to occur (Jamil et al. 2018; Nguyen et al. 2011). There are several lines of C. ternatea with different flower colours of light blue, dark blue, white and mauve which are 4–5 cm long (Fig. 1). Compounds reported to be found in the flowers are ternatin anthocyanins and various flavanol glycosides of kaempferol, quercetin and myricetin (Mukherjee et al. 2008; Kazuma et al. 2003). The leaves are pinnate with 5–7 leaflets, eliptic-oblong with a length and width range of 2.5–5.0 and 2.0–3.2 cm. They have flat linear beaked seed pods with a length range of 5–7 cm and is edible when tendered. The seed is oval in shape and has a blackish or yellowish brown colour with a length range of 4.5–7.0 mm and 3–4 mm wide. It has a taproot system with many slender lateral roots (Kosai et al. 2015; Mukherjee et al. 2008).

Fig. 1.

Fig. 1

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Clitoria ternatea flower

Nutritional analysis of C. ternatea flowers identified the percentage of protein, fibre, carbohydrate and fat to be 0.32, 2.1, 2.2 and 2.5% respectively while the moisture content was found to be 92.4%. The flower was also found to have high content of calcium (3.09 mg/g), magnesium (2.23 mg/g), potassium (1.25 mg/g), zinc (0.59 mg/g), sodium (0.14 mg/g) and iron (0.14 mg/g) (Neda et al. 2013). Several studies investigated, identified and isolated the bioactive compounds from C. ternatea flower. The anthocyanins known as ternatins are blue in colour and are acylated based on delphinidin (Fig. 2). Their structures were characterised as malonylated delphinidin 3,3′,5′-triglucosides having 3′,5′-side chains with alternative d-glucose and p-coumaric acid units at R and R1 with a total of 15 (poly) acylated delphinidin glucosides identified in all the blue petal lines namely ternatins A1-A3, B1-B4, C1-C4 and D1-D3 while some studies have identified several other delphinidin derivatives (Zakaria et al. 2018; Shen et al. 2016; Nair et al. 2015). Ternatins A1, A2, B1, B2, D1 and D2 (Fig. 3) are the six major anthocyanins present in the flowers (Mukherjee et al. 2008; Terahara et al. 1998). The flavonols (Fig. 4) identified in the petals are fourteen kaempferol, quercetin and myricetin glycosides which consist of H or OH at R1 and R2 and with H, rhamnosyl or malonyl at R3 and R4 (Mukherjee et al. 2008; Kazuma et al. 2003). Shen et al. (2016) identified various lipophilic compounds from C. ternatea being fatty acids (palmitic acid, stearic acids, petroselinic acids, linoleic acid, arachidic acid, behenic acid and phytanic acid), phytosterols (campesterol, stigmasterol, β-sitosterol and sitostanol) and tocols (α-tocopherol and γ-tocopherol). Several other components such as mome inositol, pentanal, cyclohexen, 1-methyl-4-(1-methylethylideme) and hirsutene were identified by Neda et al. (2013). In addition to the identification of various anthocyanins and flavonol glycosides, other components such as 6″‐malonylastragalin, phenylalanine, coumaroyl sucrose, tryptophan and coumaroyl glucose were determined (Zakaria et al. 2018).

Fig. 2.

Fig. 2

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Delphinidin 3-malonyl glucoside

Fig. 3.

Fig. 3

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Major anthocyanins in C. ternatea flowers a Ternatin A1, b Ternatin A2, c Ternatin B1, d Ternatin B2, e Ternatin D1, and f Ternatin D2

Fig. 4.

Fig. 4

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Flavonol glycosides

Clitoria ternatea has been used traditionally in ayurvedic medicine for various health issues. Its roots are used to treat indigestion, constipation, fever, arthritis, sore throat, skin diseases and eye ailments while its seeds are used as laxative, to treat colic and swollen joints. The traditional Cuban culture uses the decoction of the roots alone or combined with flowers to promote menstruation, induce uterine contractions as well as to treat liver and intestinal problems (Mukherjee et al. 2008; Fantz 1991). Various research studies have been done on the roots, seeds, flowers and leaves of C. ternatea. Clitoria ternatea flower is known for its potential health benefits in which several studies have shown the crude extract to have antidiabetic (Borikar et al. 2018), antioxidant (Chayaratanasin et al. 2015), antimicrobial (Leong et al. 2017) and antiproliferative/anticancer activities (Shen et al. 2016). Thus, C. ternatea flowers can be used as a natural source of antioxidants and/or a possible supplement in food or pharmaceutical industries. This paper reviews the current update on the extraction methods of C. ternatea flowers and its effect on the phytochemicals as well as the biological activities of these phytochemicals.

Extraction of phytochemicals

Extraction procedure of phytochemicals from plant materials is an important step. Various extraction procedures are available and the identification/selection of optimum parameters are important to ensure the enhancement of phytochemical yield (Azmir et al. 2013). Conventional and non-conventional extraction methods are available having respective advantages over each other thus careful selection of method should be evaluated depending on the suitability of samples and goals needed to be achieved. Prior to extraction, plant materials are usually reduced in size to increase the surface area for mixing with solvent and the samples used are either fresh, dried, grounded or powdered. Most studies on C. ternatea flowers utilized air/oven-dried, fresh flowers (Srichaikul 2018; Phrueksanan et al. 2014; Kamkaen and Wilkinson 2009) or grounded/powdered, dried flowers (Lakshan et al. 2019; López Prado et al. 2019; Mehmood et al. 2019; Pham et al. 2019; Adhikary et al. 2018; Rabeta and An Nabil 2013). Some studies utilized fresh flowers that were cut into smaller pieces, washed and stored in − 25 °C freezer and extracted within a month’s time (Chong and Gwee 2015) or freeze dried followed by grounding (Shen et al. 2016). Several conventional and non-conventional extraction methods have been used to obtain phytochemicals from C. ternatea flowers as discussed below.

Conventional extraction

Conventional extraction methods usually involve the use of different solvents with heat and/or mixing such as soxhlet extraction, maceration and hydrodistillation which though effective can be costly and require long extraction time (Wen et al. 2018; Azmir et al. 2013). Conventional extraction method is a classical method which has been widely used for the extraction of C. ternatea flower since the 1970s. Extraction studies on C. ternatea flower utilising aqueous solvent mixtures isolated and identified the structure of various phytochemicals mainly anthocyanins (Terahara et al. 1989, 1996, 1998) while other studies (Kazuma et al. 2003; Saito et al. 1985; Ranaganayaki and Singh 1979) focused on the flavonol constituents. Most studies employed extractions using aqueous solvent mixtures of ethanol or methanol rather than water alone with heating to investigate its potential bioactivities and phytochemical content while a number of studies investigated on the optimal solvent and/or extraction parameters (Table 1).

Table 1.

The extraction of phytochemicals from C. ternatea flowers

Extraction solvent Phytochemical extracted Optimal extraction condition References
Ethanol, methanol, chloroform, acetonitrile, acetone, ethyl acetate, n-butyl, water, n-hexane and ethyl ether Anthocyanins Ethanol Ludin et al. (2018)

70% ethanol

(40–80 °C)

 70 °C

70% ethanol

(pH 4–9)

 pH 4

Water

(pH 2, 7 and 10)

 Anthocyanins pH 2 Mauludifia et al. (2019)
Methanol

Anthocyanins (Ternatin A1, B2, B3, C2, D2, D3, delphinidin derivatives), kaempferol

3-neohesperidoside and quercetin 3-(2G-rhamnosylrutinoside), rutin, ellagic acid

 – Shen et al. (2016)
Ethyl acetate and hexane (1:1) Fatty acids (palmitic acid, stearic acids, petroselinic acids, linoleic acid, arachidic acid, behenic acid and phytanic acid), phytosterols (campesterol, stigmasterol, β-sitosterol and sitostanol) and tocols (α-tocopherol and γ-tocopherol) –

50% ethanol

(40–80 °C, 15–75 min, liquid–solid ratio of 10:1 to 30:1 mL/g)

 Anthocyanins 60.6 °C, 6 min, liquid–solid ratio of 23:1 mg/L Pham et al. (2019)

Water

(25–95 °C, 40–80 min liquid–solid ratio of 20:1 to 60:1)

 Extract yield 54 °C, 74 min, liquid–solid ratio of 37:1 Baskaran et al. (2019)

Water

(40–80 °C, 30–60 min liquid–solid ratio of 1 to 3 g/L)

 Phenolics 59.6 °C, 37 min, liquid–solid ratio of 3 g/L Lakshan et al. (2019)

Water

(11.7–68.3 °C, 8.78–51.21 min)

 Phenolics 40 °C, 30 min Escher et al. (2020)

40% and 50% ethanol

(with 30 min ultrasound or maceration alone for 1–7 days)

 Phenolics 50% ethanol with 30 min ultrasound Srichaikul (2018)
Flavonoids Maceration in 40% ethanol for 7 days

Water

(30–50 °C, 30–150 min, 96–240 W, liquid–solid ratio of 2–15 mL/g)

 Anthocyanins 50 °C, 150 min, liquid–solid ratio of 15 mL/g and 240 W Chong and Gwee (2015)

Water with ultrasound

(50 °C, 150 min, liquid–solid ratio of 15 mL/g and 240 W)

95% ethanol without ultrasound

(50 °C, 150 min, liquid–solid ratio of 15 mL/g)

 Water with ultrasound assistance

Water with and without ultrasound

(50 °C, 150 min, liquid–solid ratio of 15 mL/g and 240 W)

 Phenolics and flavonoids Water with ultrasound assistance Mehmood et al. (2019)

Water with heating (3 h)

Water with microwave (2 min)

 Dye (anthocyanins) Water with microwave assistance Sinha et al. (2012)
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Ludin et al. (2018) determined the extraction efficiency of anthocyanins using various solvents (high to low polarity) in which the ethanol extract was found to have the highest efficiency for anthocyanin extraction while ethyl ether extract had the lowest efficiency. The study found that solvent with higher polarity (ethanol) will have higher efficiency in extracting polar compounds such as anthocyanins.

Another study, compared and identified the differences in the phytochemical content of the flowers using hydrophilic (methanol) and hydrophobic (ethyl acetate and hexane; 1:1) extraction. The hydrophilic extract contained various anthocyanins, kaempferol and quercetin glycosides while the hydrophobic extract was composed of fatty acids, phytosterols and tocopherols showing the effect of compounds extracted depending on polarity of compounds (Shen et al. 2016).

Studies investigating the effect of pH for anthocyanin extraction found lower pH to be optimum (Mauludifia et al. 2019; Ludin et al. 2018) suggesting lower pH enhances the extraction of anthocyanins as increase in acidity changes anthocyanins to the flavium cation form (more stable) which may enhance vacuole cell wall breakage, increase solubility of pigments thus increasing the concentration of anthocyanins extracted. However, the possibility of inhibition of enzymatic oxidation of phenolics as well as the maintenance of the stability of extracted anthocyanins at low pH (Zhang et al. 2001) was suggested as a possible factor as well (Ruenroengklin et al. 2008).

Investigation of the extraction temperature for anthocyanin extraction was found to increase with an increase in temperature and 70 °C was found to be the optimum temperature (Ludin et al. 2018). The increase in extraction temperature facilitates higher extraction of anthocyanins as it increases the internal energy of the molecules which increases the diffusion and solubility of the pigments thus having a higher yield (Cacace and Mazza 2003). However, there was reduction in anthocyanin concentration at 80 °C which may be due to degradation of pigments (Ludin et al. 2018). A particular study compared the effect of extraction with water or aqueous methanol which found the latter to have a higher phenolic content. However, both extractions differed in time and temperature which may have influenced the outcome of the results thus suitable controls are required to have a fair justification and comparison of extraction variables (Rabeta and An Nabil 2013).

Several studies utilised the Response Surface Methodology (RSM) which adapts a multivariate system that fits experimental data in a statistical model by a response function to optimise the extraction of phytochemical of interest with variation of experiment parameters (temperature, extraction time, liquid–solid ratio). The utilisation of RSM enabled the identification of optimum parameters for the extraction of anthocyanins (114–132 mg/L), extract yield (23.7–53.4%) and phenolic content (594–692 mg GAE/100 g and 24–81 mg GAE/L) (Escher et al. 2020; Pham et al. 2019; Baskaran et al. 2019; Lakshan et al. 2019). Studies utilising RSM to enhance the extraction of phytochemical of interest is beneficial as it incorporates variables of interest (e.g. temperature, extraction time, liquid-solvent ratio) to be studied using a statistical approach which generates a mathematical model that optimizes extraction variables (Sreejith et al. 2014).

Non-conventional extraction methods

Non-conventional extraction methods are newer, highly efficient, safer to the environment having various advantages over conventional extraction method which include methods such ultrasound assisted extraction, microwave assisted extraction, enzyme assisted extraction, supercritical fluid extraction and pressurized liquid extraction (Wen et al. 2018; Azmir et al. 2013). To the author’s best knowledge, among the non-conventional extraction methods, only ultrasound and microwave assisted extraction have been employed to extract phytochemicals from C. ternatea flowers (Table 1).

Ultrasound assisted extraction works on the concept of acoustic waves production leading to molecular movement of solvent and sample which facilitates the leaching of organic and inorganic compounds (Herrera and De Castro 2005). Srichaikul (2018) compared the effect of short extraction time with ultrasound and long extraction time with maceration for 1–7 days for extraction efficiency of phenolic and flavonoid content using aqueous ethanol. Extraction of phenolic content was more efficient in the ultrasound assisted extraction. However, for flavonoid content, maceration in aqueous ethanol at day 7 was found to be higher than with ultrasound extraction (30 min). Although the flavonoid content was higher at day 7 in the aqueous ethanol extract without ultrasound assistance, a longer extraction time (7 days) was required to achieve this effect. A particular study utilised RSM to obtain the optimum extraction condition for anthocyanins using water by varying the extraction time, temperature, liquid–solid ratio and ultrasound frequency. The extraction using the optimized condition showed higher extraction yield of anthocyanins by 246.5% than 95% methanol without ultrasound assistance (Chong and Gwee 2015). Mehmood et al. (2019) further investigated this optimized condition by Chong and Gwee (2015) to determine the extraction efficiency of phenolic and flavonoid content from C. ternatea flowers by using water with and without ultrasound assistance. Similarly, ultrasound extraction showed a better extraction yield of phenolic and flavonoid content.

In microwave assisted extraction, electromagnetic energy is converted to heat following ionic conduction and dipole rotation mechanisms which promotes the release of solutes from the sample matrix to solvent (Alupului et al. 2012; Jain 2009). Sinha et al. (2012) compared the extraction efficiency of C. ternatea dye (anthocyanins) using conventional extraction with water and heating to microwave assisted extraction. Microwave assisted extraction was more efficient than conventional extraction having a higher dye yield. Microwave assisted extraction was also more time efficient requiring 2 min as opposed to conventional extraction method which required 3 h.

Although conventional extraction has widely been used for the extraction of these flowers, the use of non-conventional extraction method (ultrasound assistance) has shown to be superior and beneficial for the extraction of phytochemicals. Thus exploration on the use of other non-conventional extraction methods which are considered as “green techniques” would be beneficial in determining extraction efficiency of various phytochemicals. Other studies have employed the use of ultrasound, pulsed-electric field, pressurized liquid and microwave assisted extraction which were more effective than the conventional extraction method for extraction of phenolics and anthocyanins which required shorter extraction time and were also useful in preventing oxidation of compounds (Caldas et al. 2018; Corrales et al. 2008, 2009).

Biological activities

Clitoria ternatea flower contains a significant amount of phytochemicals which exhibits great antioxidant, antimicrobial, antidiabetic, anti-inflammatory and antiproliferative/anticancer properties (López Prado et al. 2019; Mahmad et al. 2018; Nair et al. 2015; Rajamanickam et al. 2015; Neda et al. 2013). Acute toxicity study using albino Wistar rats treated orally with aqueous ethanol extract (2000 mg/kg bodyweight) of the flower showed no signs of mortality or abnormality and there was no significant difference in the haematological values The extract did not display acute toxicity effects and are safe for consumption (Srichaikul 2018). Clitoria ternatea flowers can potentially be utilised as a functional food incorporated into various food products or even as a pharmaceutical supplement/drug combined with commercial drugs to improve treatment efficacy of patients.

Antioxidant activity

Oxidative stress plays a part in the development of chronic and degenerative illness such as cancer, autoimmune disorders, cardiovascular and neurodegenerative diseases. The discovery of antioxidants from natural sources is beneficial to human health (Admassu et al. 2018; Pham-Huy et al. 2008). Various studies investigated the antioxidant activity of C. ternatea flowers using antioxidant assays such as 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) radical scavenging, ferric reducing antioxidant power (FRAP), hydroxyl radical scavenging activity (HRSA), hydrogen peroxide scavenging, oxygen radical absorbance capacity (ORAC), superoxide radical scavenging activity (SRSA), ferrous ion chelating power, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical scavenging and Cu2+ reducing power assays. Clitoria ternatea flower has been shown to have potent antioxidant activity (Table 2).

Table 2.

The antioxidant activity of C. ternatea flowers from various research studies

Extract Antioxidant assay Results References
Water extract and 100% ethanol extract DPPH radical scavenging

Water extract IC50 = 1 mg/mL

Ethanol extract IC50 = 4 mg/mL

Water extract in gel formulation for inhibition of DPPH reduction = 28% at 0.5 mg/mL

 Kamkaen and Wilkinson (2009)
100% Methanol extract DPPH radical scavenging IC50 = 327 µg/mL Nithianantham et al. (2013)
Citrate buffer extract DPPH radical scavenging EC50 = 0.49 mg/mL Siti Azima et al. (2014)
FRAP 13.3 mM/g based on trolox equivalent antioxidant capacity (TEAC)
Water extract DPPH radical scavenging IC50 = 84.15 µg/mL Iamsaard et al. (2014)
FRAP 0.33 mmol/mg ascorbic equivalent
Water extract DPPH radical scavenging IC50 = 470 µg/mL Phrueksanan et al. (2014)
Oxygen radical absorbance capacity (ORAC) 17.54 μg trolox equivalents/mg extract
Reduction of free radical-induced erythrocyte hemolysis (4 h) 96.3% at 400 µg/mL
Inhibition of lipid peroxidation (4 h) 72.7% at 400 µg/mL
Water extract DPPH radical scavenging IC50 = 0.47 mg/mL Chayaratanasin et al. (2015)
Trolox equivalent antioxidant capacity (TEAC) 0.17 mg trolox/mg dried extract
Ferric reducing antioxidant power (FRAP) 0.38 mmol FeSO4/mg dried extract
HRSA IC50 = 19.2 mg/mL
SRSA IC50 = 26.3 mg/mL
Ferrous ion chelating power > 103 mg EDTA/mg dried extract

Methanol/acetone/water

(5:4:1) extract

 ORAC 490.7 μmol trolox equivalent/g extract Nair et al. (2015)
95% methanol extract DPPH radical scavenging IC50 = 95.3 µg/mL Rajamanickam et al. (2015)
Water extract ABTS radical scavenging 4.2 µM trolox equivalent/g extract Azima et al. (2017)
DPPH radical scavenging EC50 = 0.76 mg/mL
FRAP 10.9 mM trolox equivalent/g extract
ORAC 15.8 µmol trolox equivalent/g extract
Water extract ABTS radical scavenging IC50 = 42.9 µg/mL Zakaria et al. (2018)
DPPH radical scavenging IC50 = 195.5 µg/mL
Water extract with ultrasound assistance (US) and water extract with heat assistance at 50 °C (AGE) DPPH radical scavenging

US = 931.5 µg trolox equivalent/g extract

AGE = 764.3 µg trolox equivalent/g extract

 Mehmood et al. (2019)
ABTS radical scavenging

US = 13,488 µg trolox equivalent/g extract

AGE = 11,720.3 µg trolox equivalent/g extract
FRAP

US = 5834.6 µg trolox equivalent/g extract

AGE = 4195.3 µg trolox equivalent/g extract
Reducing power

US = 4539.0 µg trolox equivalent/g extract

AGE = 6154.1 µg trolox equivalent/g extract
Cu2+ reducing power

US = 12,696 µg trolox equivalent/g extract

AGE = 9549 µg trolox equivalent/g extract
Xanthine oxidase inhibition

US = 1.01 mg/mL (IC50)

AGE = 1.22 mg/mL (IC50)
Water extract, 100% and 50% methanol extract at 6 h (best condition) DPPH radical scavenging

Water extract = 11.7 mM trolox equivalent/g extract

100% methanol extract = 6.99 mM trolox equivalent/g extract

50% methanol extract = 12.2 mM trolox equivalent/g extract

 López Prado et al. (2019)
Inhibition of cholesterol oxidation

Water extract = 79.8%

100% methanol extract = 49.7%

50% methanol extract = 89.8%
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In the DPPH assay, 100% methanol extract of C. ternatea flower extract was found to be more potent than vitamin E (Nithianantham et al. 2013), whereas the water extract was found to be lower than ascorbic acid (vitamin C) (Chayaratanasin et al. 2015; Phrueksanan et al. 2014; Iamsaard et al. 2014) Some studies investigated and compared the antioxidant activity (DPPH assay) of the extracts using different solvents in which the water extract was found to be more potent than 100% ethanol extract at 15 min extraction time (Kamkaen and Wilkinson 2009). However, in another study the best extraction time was determined (6 h) for the water extract, 100% and 50% methanol extract in which the water extract and 50% methanol were found to be equally potent and had a higher activity than 100% methanol extract (López Prado et al. 2019). The optimum condition was investigated using water extract in another study with and without ultrasound at a fixed temperature and liquid-solvent ratio in which the extraction with ultrasound was found to have higher antioxidant activity (Mehmood et al. 2019). The in vitro chemical assays to measure antioxidant activity (Table 2) need to be carefully interpreted as they bear no similarity to biological systems including the absorption of antioxidants by the human body (Gengatharan et al. 2015). In a cell based study, the water extract was found to potently inhibit 2,2′-azobis-2-methyl-propanimidamide dihydrochloride (AAPH)-induced hemolysis and oxidative damage of canine erythrocytes (Phrueksanan et al. 2014). In another study, the pre-treatment of human HaCaT keratinocytes with the water extract reduced UV-induced mitochondrial DNA damage (Zakaria et al. 2018). In a randomised crossover study, acute ingestion of C. ternatea flower extract/beverage was found to have increased plasma antioxidant capacity and the effect was further enhanced when consumed together with sucrose in healthy men (Chusak et al. 2018). These studies attribute the flavonols and anthocyanins for the antioxidant activity.

Antibacterial activity

The emergence of antibiotic resistance microbes limits the effectiveness of current drugs significantly causing treatment failure of infections (Scheffler et al. 2013). In regard to this challenge, there is a need to develop alternative approaches in addition to searching for new antibacterial compounds. In vitro activity of an antimicrobial (antibacterial or antifungal) agent can be tested through various methods such as broth or agar dilution, and disc diffusion methods (Balouiri et al. 2016). Several studies investigated on the antibacterial potential of C. ternatea flowers. The methanol extract of C. ternatea flower was tested against 12 bacterial species (Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, Staphylococcus aureus, Streptococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhi, Enterobacter aerogens, Proteus mirabilis and Herbaspirillum spp.) and was found to have the most potent activity against Bacillus thuringiensis with a minimum inhibitory concentration (MIC) of 12.5 mg/mL and minimum bactericidal concentration (MBC) of 25 mg/mL with an inhibition zone of 15.7 mm using agar disc diffusion technique (Kamilla et al. 2009). In another study, the water, methanol, petroleum ether, hexane and chloroform extract of C. ternatea flower (4 mg) were tested against E. coli, K. pneumoniae, S. enteritidis, S. typhimurium and P. aeruginosa to determine its antibacterial activity. The methanol extract was found to have the highest activity when tested using agar disc diffusion technique with an inhibition zone range of 16–26 mm in E. coli, K. pneumonia and P. aeruginosa but had no activity against S. typhi and S. enteritidis. The highest zone of inhibition 26 mm was observed against K. pneumonia and P. aeruginosa (Uma et al. 2009). Leong et al. (2017) determined the antibacterial activity of anthocyanins of C. ternatea flower ethanol extract paste against B. cereus, B. subtilis, S. aureus, B. subtilis subsp. spizizenii, Proteus mirabilis, K. pneumoniae, Yersinia enterocolitica and E. coli. The extract was found to have good antibacterial activity against B. cereus, B. subtilis, S. aureus, P. mirabilis and K. pneumonia with the most potent activity against K. pneumonia with a MIC of 1.6 mg/mL and minimum lethal concentration (MLC) of 25 mg/mL while in another study, the anthocyanin fraction obtained from the ethanol extract of C. ternatea flower had the best effect against B. subtilis with a disc diffusion inhibition zone of 10 mm (Mahmad et al. 2018). The findings from these studies suggest the potential of the anthocyanins for its antibacterial activity.

Antifungal activity

A rise in resistance towards most antifungal agents in diverse pathogens which calls for the need to identify new therapeutic agents (Perfect 2016). The methanol extract of C. ternatea flower (100 mg/mL) tested against Candida albicans, Rhizopus and Penicillium spp. had the highest activity against Candida albicans with an inhibition zone of 19 mm in agar disc diffusion. However, in broth dilution method, it only had activity against Penicillium spp. and Rhizopus with similar MIC value of 0.8 mg/mL and MFC value of 1.6 mg/ml (Kamilla et al. 2009). The anthocyanin fraction obtained from the ethanol extract of C. ternatea flower tested against Fusarium sp., A. niger and Trichoderma sp. had the highest activity against Fusarium sp. with an inhibition zone of 10 mm in agar disc diffusion technique (Mahmad et al. 2018). The anthocyanins of C. ternatea flower ethanol extract paste (50 mg/mL) tested against A. niger, P. expansum and R. stolonifera only exhibited activity against P. expansum with an inhibition zone of 15.5 mm in agar disc diffusion while it had an MIC value of 12.5 mg/mL and MLC value of 25 mg/mL. The mode of action for the antifungal activity against P. expansum was investigated and found to be mediated by the alteration of morphology of P. expansum fungal hyphae which had flattened empty hyphae resulting from cell wall disruption and damage of conidiophore. The germination of P. expansum conidia was completely inhibited with suppressed conidial development (Leong et al. 2017).

Anti-inflammatory activity

The current available non-steroidal anti-inflammatory drugs (NSAIDs), including acetaminophen and aspirin are associated with side effects, particularly gastrointestinal and cardiovascular effects as they are known to affect both COX-1 and COX-2. The discovery of new or alternate strategies is needed to reduce the risks associated with NSAIDs while achieving sufficient pain relief (Brune and Patrignani 2015).

The petroleum ether extract of C. ternatea flowers was evaluated for anti-inflammatory activity using carrageenan paw edema method with healthy albino rats of either sex. The extract (200 and 400 mg/kg) significantly inhibited paw edema compared to control untreated group while in Eddy’s hot plate method, the treatment group (400 mg/kg) had significant increased reaction time (time recorded when animals licked their fore or hind paws or jump response, whichever appear first) compared to control untreated group. The study suggests the possibility of the extracts to have a protective effect against the release of prostaglandins, kinnins and other substances in carrageenan induced edema (Shyamkumar and Ishwar 2012). In another study, the anthocyanin and flavonol fraction obtained from C. ternatea flower extract (extracted in a mixture of MeOH/acetone/H2O at ratio of 5:4:1) were investigated for its anti-inflammatory potential. In the lipopolysaccharides (LPS)-induced inflammation in RAW-264.7 macrophage cells, the flavonols had mild suppression of ROS while the anthocyanins had no effect on ROS production. The anthocyanins were also found to have higher inhibition of nitric oxide production compared to the flavonols. In western blot studies, only the anthocyanins inhibited nuclear factor-κB translocation and iNOS protein expression whereas the flavonols significantly inhibited COX-2 expression but not the anthocyanins (Nair et al. 2015).

Cytotoxic and anti-proliferative/anticancer activities

Chemotherapy, radiation therapy and targeted therapy are among the approaches used for the treatment and management of cancer but they are not able to provide a permanent cure and have been associated with various side effects and toxicities (Curigliano et al. 2012). Thus, new agents that are safe, available and effective are urgently needed. Several studies investigated the anticancer potential of C. ternatea flower extracted using different solvents. The 100% petroleum ether extract (IC50 = 36 µg/mL) was found to be more potent than the 100% ethanol extract (IC50 value of 57 µg/mL) in the in vitro cytotoxic assay against Dalton’s lymphoma ascites (DLA) cells at 3 h which could be due to different phytochemical composition in both extracts. The petroleum ether extract was found to have presence of saponins, tannins, steroids and triterpernoids while the ethanol extract had flavonols only (Kumar and Bhat 2011). In another study, the water extract was more potent than the methanol extract having much lower IC50 values with activity against hormone dependent breast cancer cell line (MCF-7), non-hormone-dependent breast cancer cell line (MDA-MB-231), human ovary cancer cell line (Caov-3), and human liver cancer cell line (HepG2) at 72 h. However, the methanol extract had activity against MCF-7 and MDA-MB-231 cells only with higher IC50 values. The extracts were not toxic against the normal cell line (Hs27). The study suggests the aqueous extract to have more significant anti-proliferative activity than the methanol extract as it may have more active compounds (flavonoids) present (Neda et al. 2013). Shen et al. (2016) found the anticancer effect of the hydrophilic (100% methanol) extract to be more potent than the lipophilic (hexane:ethyl acetate, 1:1) extract on human epithelial laryngeal carcinoma (Hep-2) cell line. The potent active compounds identified in the hydrophilic extract were mainly ternatins, kaempferol and quercetin responsible for the antiproliferative effect as opposed to the lipophilic extract which constitutes of fatty acids, phytosterols and tocopherols.

Antidiabetic activity

Oral antidiabetic medications such as biguanides, meglitinide, thiazolidinedione, sulfonylureas and dipeptidyl peptidase 4 are known to be associated with various side effects (Chaudhury et al. 2017). Herbal based medications are worth exploring for potential use in the management of diabetes as they are considered to be safer and may have reduced side effects (Borikar et al. 2018). Several studies investigated the in vitro and in vivo potential of C. ternatea flower extract for antidiabetic activity. The water extract reduced the formation of fluorescent advanced glycation end products having the highest activity at day 28 (49.4% at 1 mg/mL) as well as significant reduction in fructosamine level (14.47–36.66%) in glycated bovine serum albumin. The study suggests the potential of the extract in the prevention of the formation of advanced glycation end products to be mediated through its free radical scavenging ability mainly attributed to the active compounds present being the ternatin anthocyanins, delphinidin derivatives and kaempferol (Chayaratanasin et al. 2015). In vivo study for antidiabetic activity in alloxan-induced diabetic rats (wistar albino) by Rajamanickam et al. (2015) utilising 95% methanol, ethyl acetate and chloroform extract were found to have significantly reduced blood glucose level, increased serum protein levels and restored serum albumin to normal levels. The extracts also significantly decreased serum urea, creatinine, cholesterol and triglyceride levels compared to control untreated diabetic rats. A similar trend was also observed in the in vivo studies by Borikar et al. (2018) utilising 100% methanol extract and water extract in the study by Daisy and Rajathi (2009). In a randomised crossover study, acute ingestion of C. ternatea flower extract/beverage was found to have suppressed postprandial plasma glucose and insulin levels when consumed with sucrose in healthy men (Chusak et al. 2018). Overall, these studies suggested the hypoglycemic activity may be exerted by the flavonoid principles (flavonol glycosides and anthocyanins) and alkaloids present in the extract which may involve the potentiation of insulin secretion from the β-cell or by enhancement of the transport of blood glucose from plasma to peripheral tissues.

Other biological activities of phytochemicals studied in C. ternatea flowers

Clitoria ternatea flower has shown to have potential antioxidant, antimicrobial, anticancer and antidiabetic activity. However, there are also various studies which have looked into its potential for other beneficial activities. Adhikary et al. (2018) found the 100% methanol extract of C. ternatea flower and its purified compound quercetin-3β-d-glucoside for its anti-arthritic potential in a mice model. Quercetin-3β-d-glucoside was found to be more potent than the extract to significantly reduce myeloperoxidase activity, decrease in release of pro-inflammatory cytokines, chemokines, reactive oxygen species (ROS)/reactive nitrogen species production. It also significantly reduced tumor necrosis factor α-receptor 1, toll-like receptor 2, inducible isoform of nitric oxide synthase, COX-2 and matrix metalloproteinase-2 expression.

The anti-allergy effects of C. ternatea flower extract was also found in a study by Singh et al. (2018). The 98% ethanol extract was able to attenuate histamine-induced contraction in both goat tracheal chain and isolated guinea pig ileum preparations. The extract was also found to attenuate histamine-induced dyspnoea and ovalbumin-induced changes of various inflammatory cytokines in animal models. The extract also displayed antitussive activity in sulfur dioxide- and citric acid-induced cough in experimental animals and attenuated inflammation in carrageenan and acetic acid challenged rodents. Clitoria ternatea flower extract was also found to have other beneficial effects in various other studies such as anthelmintic (Nirmal et al. 2008), larvicidal (Mathew et al. 2009), anti-aging (Zakaria et al. 2018), hepatoprotective (Nithianantham et al. 2013), testicular damage protection (Iamsaard et al. 2014), anti-adipogenesis (Chayaratanasin et al. 2019) and starch digestion (Chusak et al. 2019) activity.

Conclusion

Clitoria ternatea is a versatile plant known for its traditional application in ayurvedic medicine, food colourant and cover crop among others. Various beneficial studies have been done on this plant along the years which have found it to have many health benefits thus giving a greater insight on its potential uses. Extraction studies have been a very important step in identifying variables which influences the extraction of phytochemicals. Conventional extraction such as the use of solvent for maceration of samples has been the most common approach and shown to be efficient. However, several disadvantages have been associated with this method due to high cost, requiring long extraction time and is not environmentally friendly with the use of solvent. Thus newer studies have explored and compared extraction methods such as ultrasound/microwave assisted extraction (non-conventional method) which are not only environmentally friendly but are much more time efficient than conventional extraction methods. Thus, future studies could be geared towards the use of non-conventional extraction for C. ternatea flowers as well as exploring other environmentally safe methods such as pulsed-electric field, pressurized liquid, enzyme-assisted and pressurised liquid extraction. Findings from acute toxicity studies for C. ternatea flowers are rather encouraging as it has shown to be safe for consumption in vivo studies. Thus it is recommended for further studies to evaluate it for subchronic/chronic toxicity, histopathology as well as clinical studies to determine its effect and safety for long term consumption. Most studies have shown the phytochemicals in C. ternatea flowers, mainly anthocyanins, quercetin and kaempferol glycosides to probably be responsible for the beneficial biological effects. There are variants of anthocyanins, quercetin and kaempferol present in the flowers. Thus it would be beneficial for future studies to explore and compare the effect between anthocyanin/flavonol fractions which would help guide the potential fraction to further isolate the compound most probably responsible for the studied effect. Cytotoxicity studies have shown the extract to be more selective towards cancer cell lines than normal cells which may have less side effects in the human body. Some studies have determined the mode of action of these bioactive constituents which provides a lead in understanding the causative biological effect observed. Exploring the mechanism of action of the extract/active compounds in eliciting the biological effect are crucial to understand how it may affect/modulate certain pathways/molecular targets in the human body and are thus warranted in further studies. Numerous studies have shown C. ternatea flower for its potential antioxidant activity not only in chemical based assays but in cell based assays and in vivo studies. The consumption of C. ternatea flower extract/beverage was shown to have potential antioxidant and antihyperglycemic effects in human subjects. However, the effect was only studied in healthy subjects which may not be generalised to all population. Future studies looking on the effect on subjects with particular health conditions compared to healthy subjects are recommended to better understand its effect and determine its potential. Owing to the many benefits displayed by this flower, it is worthwhile to carry out suggested further studies to better understand the biological effects which have been reported so far as well as exploring other bioactivities. The bioactive compounds of C. ternatea flower are promising candidates for development as novel and effective pharmaceutical agents as well as applications as functional foods to promote human health and wellbeing.

Acknowledgement

This work was funded by the School of Science, Monash University Malaysia.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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