Biopharmaceutical potentials of Prosopis spp. (Mimosaceae, Leguminosa).

Prosopis is a commercially important plant genus, which has been used since ancient times, particularly for medicinal purposes. Traditionally, Paste, gum, and smoke from leaves and pods are applied for anticancer, antidiabetic, anti-inflammatory, and antimicrobial purposes. Components of Prosopis such as flavonoids, tannins, alkaloids, quinones, or phenolic compounds demonstrate potentials in various biofunctions, such as analgesic, anthelmintic, antibiotic, antiemetic, microbial antioxidant, antimalarial, antiprotozoal, antipustule, and antiulcer activities; enhancement of H+, K+, ATPases; oral disinfection; and probiotic and nutritional effects; as well as in other biopharmaceutical applications, such as binding abilities for tablet production. The compound juliflorine provides a cure in Alzheimer disease by inhibiting acetylcholine esterase at cholinergic brain synapses. Some indirect medicinal applications of Prosopis spp. are indicated, including antimosquito larvicidal activity, chemical synthesis by associated fungal or bacterial symbionts, cyanobacterial degradation products, "mesquite" honey and pollens with high antioxidant activity, etc. This review will reveal the origins, distribution, folk uses, chemical components, biological functions, and applications of different representatives of Prosopis.

1. Introduction

Small trees or shrubs that are representatives of Prosopis can be major plants component in drylands of Africa, America, and Asia. This plant is hardy, drought resistant, and fast growing [1]. The genus Prosopis accommodates 44 species, of which 40 are native to North and South Americas, three originate in Asia, and one comes from Africa. In the Americas, 28 species of this genus have been recorded, including 13 endemic species. In Taiwan, the only record of the genus was about Prosopis juliflora, found by Mr Yaiti Simada on the March 1, 1920, in Hengchun Township, Pingtung County (Specimens Database of Native Plants in Taiwan).

Chemical compounds in Prosopis spp. change certain physiological processes in the human body. Besides medicinal applications, different mesquite species have other uses. Since its wood is extremely hard and durable, and is of appealing coloration, it is used for making furniture and parquet flooring. Wood is also used for construction, as firewood, or for charcoal production [2]. Wooden chips provide mulch for gardening [3]. A beverage, known as “anapa,” is produced by mixing mesquite pods mixed in water After being fermented, it produces the alcoholic beverage “chichi” [4]. Owing to its high carbohydrate level, mesquite wood can also be used to produce bioethanol. Preliminary trials converted up to 80% of pod carbohydrates into bioethanol [3]. The aerial parts of Prosopis spp. from Argentina (Prosopis alpataco, Prosopis argentina, and Prosopis chilensis) demonstrated binding properties for DNA due to certain alkaloids. These also inhibited the antioxidant activity of β-glucosidase [5]. Medicinal values of Prosopis have been mentioned in ancient literatures [6].

An early report by Kirtikar et al [7] mentioned that all parts of Prosopis spp. are traditionally used by indigenous people for curing various ailments [8]. Water extracts of leaves and bark are traditionally used to cure mouth and throat infections, as well as bronchitis and ulcers; internal diseases including parasites and urinary diseases; and skin parasitic infections as well as dermatitis [9]. The Indian Council of Forestry Research and Education (ICFRE) reported as early as 1993 that in Asia, medicinal uses of native Prosopis species included their flowers for the prevention of miscarriage and bark extracts for the treatment of bronchitis, leucoderma, tremors, asthma, rheumatism, leprosy, and dysentery. Leaf smoke is traditionally used to cure eye infections and extracts are recommended for use against snakebite and scorpion sting [10]. Ahmad et al [11] studied the antibacterial efficiency of juliflorine and julifloricine (structures of all mentioned phytochemicals are listed in Figure 1), as well as a benzene-insoluble alkaloid fraction of P. juliflora. Similarly, P. juliflora was reported with low mean antigiardiasic activity [12]. More recently, representatives of Prosopis were studied more intensively. These studies came along with advanced scientific technologies that demonstrated several medicinal properties of these species, such as antioxidant hepatoprotective, hemolytic, anticancer, antibacterial, antifungal, antidiabetic, and anti-inflammatory activities [13].

Figure 1

Chemical structures of phytochemicals with medicinal properties present in Prosopis spp.

Alkaloids, flavonoids, terpenes, and phenolic compounds are the most important bioactive substances of Prosopis spp. [14]. Terpenes are used as insecticides, and their pharmacological properties include antibacterial, antifungal, antihelminthic, antimalarial, and molluscicidal activities [14]. Phenolic compounds from mesquite show anti-inflammatory, antitumor, anti-HIV, anti-infective, vasodilatory, antiulcerogenic analgesic, and immunostimulant activities [15]. Flavonoids have attracted interest, recently, due to the discovery of their pharmacological activities [16]. Alkaloids from mesquite are applied as analgesics and antimalarial agents. Alkaloids of Prosopis spp. also demonstrate a broad spectrum of antifungal activities against fungi such as Fusarium, Drechslera, and Alternaria. Flavonol glycosides and hydroxycinnamic acids from the pollens of P. juliflora provide antioxidants with high free radical scavenging activity [17,18]. Examples of these bioactive compounds are 3-oxo-juliprosopine and secojuliprosopinal isolated from P. juliflora [19].

Low production costs of a large amount of raw materials and straightforward integration into traditional agricultural practice provide certain advantages in the use of mesquite. This makes these plants particularly attractive in developing countries. However, the scientific validation of traditional forms of medicinal usage is still missing [20]. Most investigations of traditional medicinal applications are restricted to crude extracts with different solvents from a limited selection of representatives of Prosopis spp., with no further investigation on their bioactive compounds.

The present review summarizes the pharmaceutical potential of Prosopis spp. with respect to different diseases.

2. Biology of mesquite trees belonging to Prosopis spp

Mesquite trees have a deep-growing taproot that can even reach water tables at a depth of more than 30 m [21]. Mesquite trees exhibit species-specific differences and have a wide range of varieties. The most frequently occurring P. juliflora has a twisted stem with branches armed with strong thorns. Its leaves are bipinnate, the pale yellow flowers are arranged in spikes, it has flattened fruits with a solid epicarp, and the curved, pulpy mesocarp is sweet with several seeds [2,3].

P. juliflora is adapted to warm and dry tropical climates. It can grow in areas with an annual rainfall of only 250–600 mm. It is fast growing with a well-meshed deep-growing root system, as studied by Yoda et al [22]. P. juliflora can grow in extreme situations such as in rocky and saline soils. It is adapted to drought, and its leaves are avoided by most herbivores in agriculture. It is suitable for reforesting wastelands [23]. Some authors claim that P. juliflora also provides important ecosystem services related to the soil physical structure and nutrient cycling, compared with conventional plants in conventional monoculture. It recovers after enduring frost and moderate fire. It is claimed that after sprouting back in early spring, P. juliflora would regain its original size within less than 1 year [24].

Several Prosopis species can endure high temperatures and low rainfall, as well as saline, infertile, and even alkaline soils [3,25]. Whereas other congeners fail to withstand prolonged drought periods, P. juliflora endures those harsh times. Its seedlings survive drought by protecting young leaves with their folded cotyledons.

3. Medicinal applications

3.1. Antibacterial activity

Microbial antibiotic resistance is a matter of increasing concern. This calls for novel approaches in obtaining antimicrobial activity to treat infectious diseases in humans as well as in agricultural animals and plants. According to Ahmad et al [11], the compound juliflorine, which is generally synthesized within the genus Prosopis, provides protection against some human pathogenic bacteria, such as Corynebacterium diphtheria var. mitis, Corynebacterium hofmanni, Bacillus subtilis, Staphylococcus aureus, and Streptococcus pyogenes (see Table 1). Interestingly, juliflorine offered substantial antibiotic efficacy even against Streptococcus faecalis, which is resistant to most antibiotics [26,27]. Compared with the antibiotics streptomycin and penicillin, ethanol extracts of Prosopis spp. provide antibacterial activity against various pathogenic bacteria (see Table 1) [28]. Among human pathogenic bacteria, Pseudomonas aeruginosa provides one of the most threatening multidrug-resistant microbes, which has a multidrug efflux system containing at least 10 separated efflux pump system genes [29]. The minimal inhibitory concentrations of several unrelated antibiotics are increased by this efflux pump mechanism [30,31]. The crude methanol extracts of P. juliflora showed antibacterial activity against S. aureus and Escherichia coli [32]. The compounds myoinositol-4C-methyl and N-β-chloropropionyltryptamine (Figure 1) are particularly responsible for this activity [33]. In comparison to extracts of other plant parts, the extract of P. juliflora pods shows higher activity against Gram-positive bacteria, such as S. aureus [34].

Table 1

Traditional usage of Prosopis spp. in herbal medicine.

Species	Parts used	Treatment	Reference
P. cineraria	Leaf smokeLeaf extractGum	Treatment for eye infection, snakebite, and scorpion sting	[10]
P. cineraria	Flower	Safeguarding pregnant women against miscarriage	[89]
P. cineraria	Pods	Efficacious in rheumatism	[90]
P. juliflora	Leaf juice	Folk remedies for cancerous conditions	[91]
P. juliflora	Pods and Leaves	Remedy for catarrh cold, diarrhea, dysentery, excrescences, eye infections, flu, head cold, hoarseness, inflammation, itch, measles, pink eye, stomachache, sore throat, and wounds	[92]
P. juliflora	Leaf tea	Remedy for sore throat	[93]
P. grandulosa	Paste of leaves and fruit	Relieving pain associated with bone fracture in animals	[94]

Different parts of P. juliflora provide different alkaloids, tannins, phenols, flavonoids, terpenes, and steroids [35,36]. Furthermore, ethanolic leaf and root extracts of P. juliflora showed antibacterial abilities against Gram-negative bacteria that were otherwise resistant to antibiotics such as minocycline, chloramphenicol, and erythromycin [37]. It was also noted that alkaloids, saponins, and tannins were likely candidates for this activity [38]. Plant diseases caused by the bacteria Agrobacterium rhizogenes and Xanthomonas campestris were shown to be cured by aqueous extracts of P. juliflora [39].

3.2. Antifungal activity

A severe deadly fungal disease harmful to patients infected by HIV/AIDS is caused by the fungal species Cryptococcus meningitis. Cryptococcus neoformans and Cryptococcus gattii are also involved [40]. Alkaloid-rich fractions of P. juliflora leaves inhibit the growth of C. neoformans, and the compounds zerumbone and cassine (Figure 1) exert high antifungal activity against this fungal species [41]. Plants’ defense mechanisms vary depending on the fungal pathogens they face [42]. Methanol extracts of P. juliflora leaves have substantial anti-fungal activity against mycelial growth of the fungus Colletotrichum musae, causing the most serious postharvested fungal disease in banana [43]. As shown by other experiments, the methanol extract of leaves is thermostable, showing tolerance of plant extracts to higher temperatures. This fraction shows considerable antifungal activity against seed-borne fungal pathogens, such as Aspergillus candidus, Aspergillus columnaris, Aspergillus flavipes, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aspergillus ochraceus, and Aspergillus tamarii. Methanol and aqueous extracts of P. juliflora also show anti-fungal activities, especially against A. niger [44,45] and A. fumigatus [39], respectively. A study of tobacco plant infections caused by the fungal pathogen Alternaria alternata demonstrated the antifungal efficacy of P. juliflora leaf extracts [46]. Aqueous extracts of P. juliflora showed higher activity than synthetic fungicides.

3.3. Antiprotozoal activity

Eukaryotic protist infections are causing serious impacts on people, especially immune-compromised people, worldwide [47]. Methanol extracts of P. juliflora have high antiplasmodial activity against the malaria-causing protist Plasmodium falciparum and also antiflagellate activity against the vector of the Chagas disease, Trypanosoma cruzi [48]. Petroleum-ether extracts of P. juliflora leaves were assessed for intestinal, performing antigiardial, and amoebicidal activities and activity against pathogenic protists [49]. A study demonstrated high activity against Entamoeba histolytica, with 71.97% mortality after 72-hour exposure to the extract at a concentration of 1000 ppm. The study also showed high antibiotic activity against the protist Giardia lambila at 500 ppm.

3.4. Antioxidant potentials of Prosopis spp

Alkaloids from Prosopis spp. have a strong ability to capture free oxygen radicals [5]. Free radicals cause oxidative damage. Several other studies confirmed that P. juliflora have substantial antioxidant potential [28]. According to the research of Lakshmibai et al [50], ethanol extracts of P. juliflora leaves containing phytochemical alkaloids were able to scavenge free radicals. Siahpoosh and Mehrpeyma [51] demonstrated that polyphenol compounds from the bark of P. juliflora showed considerable dose-dependent antioxidant and free radical scavenging activities [45].

3.5. Anti-inflammatory activity

Anti-inflammatory activity of P. juliflora was demonstrated by carrageenan- and histamine-induced paw edema in rats [19]. Carrageenan is an organic nitrogenous compound (polysaccharide and histamine) used by Sivakumar et al [19] to induce inflammation in rats. Assays of carrageenan-induced paw edema and second histamine-induced paw edema indicated potent inhibition of inflammation by P. juliflora. Involvement of prostaglandins in the second phase of inflammation makes the formulation of anti-inflammatory drugs challenging; however, according to the experiments of Sivakumar et al [19], methanol extracts of P. juliflora bark inhibited carrageen-induced inflammation in rats by blocking prostaglandins. Moreover, ethanol extracts of the leaves of P. juliflora have substantial anti-inflammatory potency [52].

3.6. Oral disinfection

To overcome oral and periodontal infections, aqueous extracts of P. juliflora leaves can be used to eradicate bacterial infections from the periodontal space and oral cavity. Its efficacy is comparable with that of commercial mouth rinse containing synthetic compounds such as chlorhexidine gluconate, sorbitol, alcohol, n-propanol, eucalyptol, methyl salicylate, thymol, and sodium benzoate [53].

3.7. Antipustule activity

In patients with inflammatory skin disorders, such as atopic dermatitis, increasing severity of the disease strongly correlates with a decrease in microbial diversity and an increase in staphylococci. Metabolites of P. juliflora also possess anti-pustule activity. Acetone extracts of leaves, for example, can inhibit the growth of Staphylococcus sp. effectively by providing antipustule plant metabolites [54].

3.8. Antiulcer activity

Compounds such as alkaloids, flavonoids, tannins, anthraquinones, and quinones from P. juliflora have been explored as therapeutic drugs against ulcer. They can inhibit the growth of the ulcer-causing bacterium Helicobacter pylori through a mechanism where H+, K+, and ATPase are inhibited in combination with antioxidant activity. Ethanol extracts of P. juliflora exhibit a higher potential for ulcer reduction in rats than the normally available drug ranitidine [55]. Gobinath et al [56] showed the safe antipyretic activity of P. juliflora crude ethanol extracts.

3.9. Antiemetic activity

Crude methanol extracts of the leaves of Prosopis cineraria and P. juliflora (as well as other plant family representatives Adenanthera pavonina and Peltoforum roxburghii) were evaluated for their antiemetic activity in a study by Hasan et al [57]. The authors assayed the antiemetic activity by calculating the mean decrease in the number of reverse movements of the stomach and esophagus without vomiting. All extracts showed antiemetic activity when compared with the standard drug chlorpromazine at the same dose. Among all extracts, P. juliflora showed the highest antiemetic activity [57].

3.10. Role in antidiabetic activity

P. juliflora contains 24-methylencycloartan-3-one (Figure 1), which can safely be used to treat diabetes mellitus instead of using insulin [58]. This compound is contained in the oil of P. juliflora pods. Additionally, it has a good hypoglycemic effect in the screening assay of alloxan inducing fasted diabetic rabbits. The compound 24-methylencycloartan-3-one shows no cytotoxicity to red mammalian blood cells [6].

3.11. P. juliflora in cancer prevention and therapy

For the In vitro antitumor potentials, total alkaloid extractions from leaves of P. juliflora contain higher concentration-dependent cytotoxic effects on cancer cells than those of normal cells. This was indicated by effective cytotoxic activity against human T-cell leukemia cells [59].

Compounds from flowers of P. juliflora show anti-proliferative activity against mitotic cell divisions via chromosome aberrations. Antiproliferative activity is demonstrated by the root cells of the onion plant Allium cepa. A new compound found in 2012 from P. juliflora flowers provides a good spindle inhibitor which can proceed the clastogenic effects in A. cepa cells [60]. This compound induces different mutations such as chromosomal aberrations, fragmentations, and C-mitotic effects.

3.12. P. juliflora in Alzheimer therapy

Alzheimer disease is a neurodegenerative disorder, such as dementia, which most occurs in older people. Cholinergic brain synapses and neuromuscular junctions are affected. The World Health Organization declared dementia as a priority condition through the Mental Health Gap Action Program in 2008 [61]. Acetyl cholinesterase plays an important role in the functioning of neuronal synapses. Acetyl cholinesterase hydrolyzes cationic neurotransmitters that terminate impulse transmission [62,63] (Figure 2).

Figure 2

Mechanisms of action of juliflorine from Prosopis spp.

P. juliflora contains specific alkaloids that affect neuro-degenerative diseases. The alkaloid juliflorine from P. juliflora can inhibit acetyl cholinesterase in a concentration-dependent manner. Choudhary et al [64] proved that juliflorine plays a potent synergy to acetylcholinesterase by producing different forces such as hydrophobic contacts and hydrogen bond between juliflorine and amino acid residues of the aromatic gorge in acetylcholinesterase (Figure 2). Amino acid residues, in particular Tyr70, Asp72, Tyr121, Trp279, and Tyr334, belonging to the anionic and peripheral sites of acetylcholinesterase are involved. Juliflorine also showed dose-dependent spasmolytic and Ca2+ channel-blocking activity in jejunum preparations of rabbits [63].

4. Veterinary medical applications

4.1. Anthelmintic activity

Nematode infections of the gastrointestinum cause mortality, loss of nutrient absorbance efficiency, weakness, and retarded growth in ruminant livestock. Even the death of animals due to nematode infections is common in tropical regions where control programs are solely based on the use of synthetic anthelmintica. These are of high costs to poor farmers and of concern with respect to the presence of residues in food and the environment. Alternative control methods are thus required. Analysis of nematode egg hatching assays demonstrated striking differences in the bioactivities of the ethanol extracts [65] of P. juliflora roots and leaves.

The anthelmintics with an effective potential for livestock are provided by aqueous leaf extracts of P. juliflora and roots of Entada leptostachya powder. The herbal drugs mixture contains compounds as alkaloids, steroids, phenolic compounds, tannins, flavonoids, and saponins [66]. This could provide an alternative to the use of chemical drugs for deworming applications. The encapsulated ethanolic extracts of P. juliflora leaves display ovicidal potential against Haemonchus contortus, a highly pathogenic nematode parasite of small ruminants [67]. The leaves inhibit egg hatching of H. contortus. Nematode egg hatching inhibition may offer a suitable bioassay for the estimation of compound efficacies.

5. Indirect medicinal effects of P. juliflora

5.1. Antimosquito activity

Mosquito control by synthetic insecticides is creating environmental problems worldwide. Larvicidal activity of acetone extracts of P. juliflora leaves provide substantial mortality against Anopheles stephensi mosquito larvae [68]. Representatives of the genus Anopheles are major vectors of malaria. Its application would provide an ecofriendly approach to mosquito control. Along with its larvicidal activity, it also shows considerable activity against the adults of the mosquito A. stephensi [69].

5.2. Biomedical applications of products associated with or processed from P. juliflora

A high amount of antimicrobial compounds from P. juliflora could be mainly due to the capability of fungal endophytes to provide benefits to its host plant [70]. A fungal endophyte from the leaves of P. juliflora shows antimicrobial potential against plant and human pathogenic bacteria and fungi. The ethyl acetate extracts of fungal endophytes isolated from the leaves of P. juliflora reveal activity against pathogenic bacteria and other fungi [71]. The endophyte Paecilomyces lilacinus, for example, exerts significant antifungal activity against the fungus Colletotrichum gloeosporioides, which in turn shows antibacterial activity.

5.3. Monofloral honey and pollens from “mesquite”

Ethanol crude extracts of honeybee-collected pollens of P. juliflora have significant inhibitory activity against lipid peroxidation. The flavonols present in monospecific mesquite honeybee-collected pollens are at least partly responsible for its antioxidant capacity. It provides inhibition against lipid peroxidation in rodents [71].

5.4. Medicinal substances of cyanobacteria-degraded products of P. juliflora

Ethanol extracts of P. juliflora degraded by the cyanobacterium Oscilatoria laetevirens, with abundant alkaloids, flavonoids, terpenoids, and steroids, provide potent free oxygen radical scavenging activity [72]. P. juliflora extracts also provide anti-oxidants that include alphatocopherol and probucol (Figure 1). Probucol shows considerable antioxidant activity against inflammatory diseases. Degraded ethanol extracts of P. juliflora can have considerable potential for pharmacological applications [73].

5.5. Mucilage of P. juliflora as tablet binder

The hydrophilic mucilage from the seeds of P. juliflora was studied for its potential of mucilage binding in tablet formulations [74]. In this study, granules were prepared by a wet granulation technique. The granules had excellent flow properties. Tablets prepared using 8% and 10% of mucilage were harder than other formulations and showed drug release over a period of 5 hours.

5.6. Probiotic and nutritional values

Mesquite pods have a high nutritional value. Astudillo et al [75] described the pod composition of different mesquite representatives from Chile as rather variable. In their study, Prosopis spp. contained about 16–41% of total sugars, 10–15% DM (dry matter) protein, and 20–30% DM crude fiber. Fresh leaves contain about 17–20% protein and 22% crude fiber. They are rich in lysine but deficient in methionine and cysteine. The hay contains about 14% protein. Seeds contain up to 30–40% protein and much less fiber (3–7%) than the pods. Pod husks are rich in crude fiber (54% DM) and poor in protein (4% DM) [75]. Pods from India and Africa have less carbohydrate but more fiber than pods from Brazil and Peru. The sugar content makes them palatable to animals in the agricultural industry.

6. Conclusion

The plant genus Prosopis has important applications in medicinal products for human use as well as in veterinary medicine (Table 2), include antidiabetic, anti-inflammatory, anticancer, and antimicrobial activities. Compounds from this taxon demonstrate antibiotic activity against microbial pathogens and enzyme inhibition activities, and show potential in other pharmaceutical applications, such as binding abilities for tablet production, inhibition of H+, K+, ATPase of H. pylori, and acetylcholine esterase inhibiting activity. Especially, the components, flavonoids, tannins, alkaloids, quinones, or phenolic compounds, demonstrate potentials in various biofunctions.

Table 2

Medicinal effects of bioactive substances from parts of the Prosopis spp (Leguminosae, Mimosaceae).

Prosopis species	Parts	Medicinal effects	Bioactive components	References
P. juliflora	Leaves	Inhibition of acetyl cholinesterase Blocking calcium channels	Juliflorine	[64]
	Leaves, pods	Decreasing gas production during ruminal digestion	JuliprosopineProsoflorineJuliprosine	[76]
	Fruits	Activity against lung carcinoma	Patulitrin	[77]
	Heart wood	Antioxidant activity	(−)-Mesquitol	[78]
	Leaves	Inhibition of drug-resistant fungi	Piperidine alkaloids	[41]
		Glial cell activation	Alkaloids	[79]
	Seeds, leaves	Inhibition of H+, K+, ATPase of H. pylori	Alkaloids, flavonoids, tannins, anthraquinones, and quinones	[56]
P. cineraria	Pods	Antioxidant activity	TriterpenoidsProsophyline	[80]
		Cyclooxygenase enzyme inhibition activity	Linoleic acid
Prosopis africana	Leaves, roots and Stem	Antitripanosomal activity	Tannins	[81]
	Stem bark	Anti-inflammatory activity	Flavonoids	[75]
	Stem bark	Antibacterial activity (Mycobacterium aurum, S. aureus)Breast cancer cell line inhibition	7′, 3′, 4-Trihydroxy-3-methoxyflavoneDehydroabietic acid	[82]
P. chilensis	Leaves	DNA binding activity	β-phenethylamineTryptamine	[75]
Prosopis glandulosa var. glandulosa	Leaves	Anti-infectiveAntiparasiticAntiparasiticAntimicrobial activity	IndolizidineJuliprosopine	[83]
Prosopis flexuosa	Aerial parts	Antioxidant activity	Catechin	[5]
Prosopis tamarugo	Leaves			[75]
Prosopis alpha	Leaves, pods			[84]
Prosopis alba	Pod flour		Schaftoside	[85]
Prosopis nigra
P. alpataco	Seed	Antibacterial activity	Pentacyclic triterpenes	[86]
Prosopis denudans
Prosopis pallida	Fruit	Antihyperglycemia	Phenolic compound	[87]
		Antihypertension
P. cineraria	Whole plant	Anticonvulsant activity	Alkaloids	[88]